Immunotherapy and diagnosis of mucormycosis using coth

ABSTRACT

The invention provided Mucorales CotH antibodies, polypeptides, encoding nucleic acid molecules, and uses thereof. The Mucorales CotH antibodies, polypeptides and encoding nucleic acids disclosed herein can be advantageously used to diagnose, treat or prevent fungal conditions, in particular mucormycosis.

RELATED PATENT APPLICATIONS

This patent application is a continuation of, and claims the benefit of,U.S. patent application Ser. No. 15/043,025 filed Feb. 12, 2016, nowpending and entitled IMMUNOTHERAPY AND DIAGNOSIS OF MUCORMYCOSIS USINGCotH, naming Ashraf S. Ibrahim, Mingfu Liu, Teklegiorgis Ghebremariam,Yue Fu, John E. Edwards and Scott Filler as inventors, and designated byAttorney Docket No. 022098-0443421; which claims the benefit of, U.S.patent application Ser. No. 13/620,563 filed Sep. 14, 2012, now U.S.Pat. No. 9,279,002, entitled IMMUNOTHERAPY AND DIAGNOSIS OF MUCORMYCOSISUSING CotH, naming Ashraf S. Ibrahim, Mingfu Liu, TeklegiorgisGhebremariam, Yue Fu, John E. Edwards and Scott Filler as inventors, anddesignated by Attorney Docket No. 022098-0436276; which claims thebenefit of U.S. Provisional Patent Application No. 61/535,257, filedSep. 15, 2011, entitled IMMUNOTHERAPY AND DIAGNOSIS OF MUCORMYCOSISUSING CotH. The entire content of the foregoing applications isincorporated herein by reference, including all text, tables anddrawings.

GOVERNMENT SUPPORT

This invention was made with government support under NIH grant numbers011671 and 013377 awarded by NIAID. The government has certain rights inthe invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 11, 2018, isnamed SeqListingLABioMed0502103.txt and is 179 KB (184,155 bytes) insize.

BACKGROUND OF THE INVENTION

The present invention relates generally to compositions and methods fordetecting, treating and preventing infectious diseases in a patient, andmore specifically to compositions and methods that target specificproteins or nucleic acids unique to fungi that cause mucormycosis.

About 180 of the 250,000 known fungal species are recognized to causedisease (mycosis) in man and animal. Some fungi can establish aninfection in all exposed subjects, e.g., the systemic pathogensHistoplasma capsulatum and Coccidioides immitis. Others, such asCandida, Aspergillus species and Zygomycetes are opportunist pathogenswhich ordinarily cause disease only in a compromised host. Fungi of theclass Zygomycetes, order Mucorales, can cause Mucormycosis, apotentially deadly fungal infection in humans. Fungi belonging to theorder Mucorales are distributed into at least six families, all of whichcan cause mucormycosis (Ibrahim et al. Zygomycosis, p. 241-251, In W. E.Dismukes, P. G. Pappas, and J. D. Sobel (ed.), Clinical Mycology, OxfordUniversity Press, New York (2003); Kwon-Chung, K. J., and J. E. Bennett,Mucormycosis, p. 524-559, Medical Mycology, Lea & Febiger, Philadelphia(1992), and Ribes et al. Zygomycetes in Human Disease, Clin MicrobiolRev 13:236-301 (2000)). However, fungi belonging to the familyMucoraceae, and specifically the species Rhizopus oryzae (Rhizopusarrhizus), are by far the most common cause of infection (Ribes et al.,supra). Increasing cases of mucormycosis have also been reported due toinfection with Cunninghamella spp. in the Cunninghamellaceae family(Cohen-Abbo et al., Clinical Infectious Diseases 17:173-77 (1993);Kontoyianis et al., Clinical Infectious Diseases 18:925-28 (1994);Kwon-Chung et al., American Journal of Clinical Pathology 64:544-48(1975), and Ventura et al., Cancer 58:1534-36 (1986)). The remainingfour families of the Mucorales order are less frequent causes of disease(Bearer et al., Journal of Clinical Microbiology 32:1823-24 (1994);Kamalam and Thambiah, Sabouraudia 18:19-20 (1980); Kemna et al., Journalof Clinical Microbiology 32:843-45 (1994); Lye et al., Pathology28:364-65 (1996), and Ribes et al., (supra)).

The agents of mucormycosis almost uniformly affect immunocompromisedhosts (Spellberg et al., Clin. Microbiol. Rev. 18:556-69 (2005)). Themajor risk factors for mucormycosis include uncontrolled diabetesmellitus in ketoacidosis known as diabetes ketoacidosis (DKA), otherforms of metabolic acidosis, treatment with corticosteroids, organ orbone marrow transplantation, neutropenia, trauma and burns, malignanthematological disorders, and deferoxamine chelation-therapy in subjectsreceiving hemodialysis.

Recent reports have demonstrated a striking increase in the number ofreported cases of mucormycosis over the last two decades (Gleissner etal., Leuk. Lymphoma 45(7):1351-60 (2004)). There has also been analarming rise in the incidence of mucormycosis at major transplantcenters. For example, at the Fred Hutchinson Cancer Center, Man et al.have described a greater than doubling in the number of cases from1985-1989 to 1995-1999 (Man et al., Clin. Infect. Dis. 34(7):909-17(2002)). Similarly, Kontoyiannis et al. have described a greater thandoubling in the incidence of mucormycosis in transplant subjects over asimilar time-span (Kontoyiannis et al, Clin. Infect. Dis. 30(6):851-6(2000)). Given the increasing prevalence of diabetes, cancer, and organtransplantation in the aging United States population, the rise inincidence of mucormycosis is anticipated to continue unabated for theforeseeable future.

Therefore, there exists a need for compounds and methods that can reducethe risk of mucormycosis pathogenesis and provide effective therapieswithout adverse effects. The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF INVENTION

In accordance with the present invention, there are provided MucoralesCotH polypeptides and encoding nucleic acid molecules. The MucoralesCotH polypeptides and encoding nucleic acids can be advantageously usedto diagnose, treat or prevent fungal conditions, in particularmucormycosis. Furthermore, the Mucorales CotH polypeptides and encodingnucleic acids are useful to generate or screen for agents that can alterMucorales CotH activity or expression, which can further be used totreat or prevent fungal conditions.

The invention also provides vectors containing Mucorales CotH nucleicacids, host cells containing such vectors, Mucorales CotH anti-sensenucleic acids and related compositions. The invention additionallyprovides Mucorales CotH oligonucleotides that can be used to hybridizeto or amplify a Mucorales CotH nucleic acid. Anti-Mucorales CotHspecific antibodies are also provided. Further provided are kitscontaining Mucorales CotH nucleic acids or Mucorales CotH specificantibodies. Such kits and reagents can be used to diagnose fungalinfection cause by Mucorales organisms. Also provided are pharmaceuticaland vaccine compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a FAR-western blot of four open reading frames (ORF)predicted to be cell surface proteins.

FIG. 2 shows GRP78 binds to germlings but not to spores of R. oryzae.

FIG. 3 shows expression of the four glycosylphosphatidylinisotol (GPI)anchored proteins predicted to act as ligands to GRP78.

FIG. 4, panels A and B, show expression of CotH genes in R. oryzaegermlings incubated with endothelial cells.

FIG. 5 shows homology of the 4 putative GPI-anchored proteins to eachother and the number of predicted N- and O-glycosylation sites in CotH3.

FIG. 6, panel A demonstrates the ability of R. oryzae to adhere to humanumbilical endothelial cells but not plastic. Further, Saccharomycescerevisiae doesn't adhere to endothelial cells. Panel B, shows R. oryzaeCotH2 and CotH3 enabling S. cerevisiae to bind endothelial cellsexpressing GRP78.

FIG. 7 shows CotH3 is conserved among various Mucorales including R.oryzae 99-880, R. oryzae 99-892, Mucor sp., Lichtheimia corymbifera,Cunninghamella bertholetiae and R. microsporus.

FIG. 8, panels A and B, show S. cerevisiae expressing CotH2 or CotH3adhere to and invades endothelial cells or CHO cells overexpressingGRP78 but not CHO parent cells.

FIG. 9 shows the amino acid sequence (SEQ ID NO. 1) and the nucleic acidcoding sequence (SEQ ID NO. 2) of CotH1 from R. oryzae.

FIG. 10 shows the amino acid sequence (SEQ ID NO. 3) and the nucleicacid coding sequence (SEQ ID NO. 4) of CotH2 from R. oryzae.

FIG. 11 shows the amino acid sequence (SEQ ID NO. 5) and the nucleicacid coding sequence (SEQ ID NO. 6) of CotH3 from R. oryzae.

FIG. 12 shows the amino acid sequence (SEQ ID NO. 7) and the nucleicacid coding sequence (SEQ ID NO. 8) of RO3G_16295 from R. oryzae.

FIG. 13 shows detection of CotH3 in sheep's blood spiked with R. oryzaeby PCR using oligonucleotide primers having the nucleic acid sequence ofSEQ ID NO: 33 and SEQ ID NO: 34.

FIG. 14 shows detection of CotH3 in sheep's blood spiked with Mucor sp.or Lichtheimia corymbifera by PCR using oligonucleotide primers havingthe nucleic acid sequence of SEQ ID NO: 33 and SEQ ID NO: 34.

FIG. 15 shows detection of CotH3 in sheep's blood spiked withCunninghamella bertholetiae or R. microsporus by PCR usingoligonucleotide primers having the nucleic acid sequence of SEQ ID NO:33 and SEQ ID NO: 34.

FIG. 16 shows no detection of CotH3 in sheep's blood spiked withAspergillus fumigates or Candida albicans by PCR using oligonucleotideprimers having the nucleic acid sequence of SEQ ID NO: 33 and SEQ ID NO:34.

FIG. 17 shows the highest homology of any of the CotH predicted proteins(in this case CotH3 [RO3G_11882] SEQ ID NO: 5) with an amino acidsequence of a protein from Stigmatella aurantiaca (ZP_01460584) (SEQ IDNO: 65).

FIG. 18 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Talaromyces stipitatus ATCC10500 (EED23986) protein (SEQ ID NO: 67).

FIG. 19 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Penicillium marneffei ATCC18224 (XP_002144175) protein (SEQ ID NO: 68).

FIG. 20 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Aspergillus niger(XP_001392236) protein (SEQ ID NO: 69).

FIG. 21 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Aspergillus nidulans(XP_658934) protein (SEQ ID NO: 70).

FIG. 22 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Ustilago maydis (XP_760027)protein (SEQ ID NO: 71).

FIG. 23 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Coccidioides immitis(XP_001243211) protein (SEQ ID NO: 72).

FIG. 24 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Neurospora crassa (XP_956792)protein (SEQ ID NO: 73).

FIG. 25 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Cryptococcus neoformans(XP_775558) protein (SEQ ID NO: 74).

FIG. 26 shows an amino acid sequence alignment between the RO3G_16295protein from R. oryzae (SEQ ID NO: 66) and Streptomyces lividans(EFD65170) protein (SEQ ID NO: 75).

FIG. 27 shows a nucleic acid sequence of CotH3 from Rhizopus oryzae99-880 (including introns from Data base) (SEQ ID NO: 9).

FIG. 28 shows a nucleic acid sequence of CotH3 from Rhizopus oryzae99-880 (exon only from Data base) (SEQ ID NO: 10).

FIG. 29 shows an amino acid sequence of CotH3 from Rhizopus oryzae99-880 (predicted amino acids) (SEQ ID NO: 11).

FIG. 30 shows a nucleic acid sequence of CotH3 from Rhizopus oryzae99-880 (including introns from sequenced data) (SEQ ID NO: 12).

FIG. 31 shows a nucleic acid sequence of CotH3 from Rhizopus oryzae99-892 (exons only from Sequenced data) (SEQ ID NO: 13).

FIG. 32 shows a nucleic acid sequence of CotH3 from Rhizopus oryzae99-892 (including intron from Sequenced data) (SEQ ID NO: 14).

FIG. 33 shows the predicted amino acid sequence of CotH3 from R. oryzae99-892 (excluding introns) (SEQ ID NO: 15).

FIG. 34 shows the predicted amino acid sequence of CotH3 from R. oryzae99-892 (including introns) (SEQ ID NO: 16).

FIG. 35 shows a nucleic acid sequence of CotH3 sequence from Mucor sp.99-932 (exons only from sequenced data) (SEQ ID NO: 17).

FIG. 36 shows the predicted amino acid sequence of CotH3 from Mucor99-932 (AA exons only) (SEQ ID NO: 18).

FIG. 37 shows a nucleic acid sequence of CotH3 from Mucor 99-932(including introns) (SEQ ID NO: 19).

FIG. 38 shows the predicted amino acid sequence of CotH3 from Mucor99-932 (including introns) (SEQ ID NO: 20).

FIG. 39 shows a nucleic acid sequence of CotH3 from Lichtheimiacorymbifera (exons only from sequenced data) (SEQ ID NO: 21).

FIG. 40 shows the predicted amino acid sequence of CotH3 fromLichtheimia corymbifera (exons only from sequenced data) (SEQ ID NO:22).

FIG. 41 shows a nucleic acid sequence of CotH3 from Lichtheimiacorymbifera (including introns) (SEQ ID NO: 23).

FIG. 42 shows the predicted amino acid sequence of CotH3 fromLichtheimia corymbifera (including introns) (SEQ ID NO: 24).

FIG. 43 shows a nucleic acid sequence of CotH3 sequence fromCunninghamella bertholetiae (exons only) (SEQ ID NO: 25).

FIG. 44 shows the predicted amino acid sequence of CotH3 fromCunninghamella bertholetiae (exons only from sequenced data) (SEQ ID NO:26).

FIG. 45 shows a nucleic acid sequence of CotH3 from Cunninghamellabertholetiae (including introns) (SEQ ID NO: 27).

FIG. 46 shows the predicted amino acid sequence of CotH3 fromCunninghamella bertholetiae (including introns from sequenced data) (SEQID NO: 28).

FIG. 47 shows a nucleic acid sequence of CotH3 from R. microsporus(exons only from sequenced data) (SEQ ID NO: 29).

FIG. 48 shows the predicted amino acid sequence of CotH3 from R.microsporus (exons only from sequenced data) (SEQ ID NO: 30).

FIG. 49 shows a nucleic acid sequence of CotH3 from R. microsporus(including introns, has only one intron) (SEQ ID NO: 31).

FIG. 50 shows the predicted amino acid sequence of CotH3 from R.microsporus (including introns) (SEQ ID NO: 32).

FIG. 51, panels A and B, show a FAR western blot of R. oryzae surfaceproteins that bound to GRP78 (Panel A) and a dendrogram showing theclose identity between CotH2 and CotH3 predicated proteins and thedivergence of the CotH proteins from the fourth identified ORF widelypresent in fungi without an identified function (i.e. RO3G_16295) (PanelB).

FIG. 52, panels A-C, show expression of CotH genes and RO3G_16295 inresponse to germination and to host cell interaction. All CotH geneswere expressed in resting spores but only CotH3 was expressed ingermlings of R. oryzae grown in YPD at 37° C. (Panel A). Exposure of R.oryzae germlings to endothelial cells induced expression of only CotH2and CotH3 genes as determined by RT-PCR (Panel B). Quantification ofgene expression of CotH genes in R. oryzae germlings on endothelialcells by qRT-PCR demonstrated 16 and 4 fold increase in expression ofCotH3 and CotH2 relative to the non expressed CotH1, respectively.RO3G_16295 was not expressed under any of the conditions tested. *P<0.001 vs. CotH1 expression and **P<0.001 vs. CotH1 and CotH2expression, by Wilcoxon Rank Sum test (Panel C). N=9 from threeindependent experiments.

FIG. 53 shows antibodies raised against peptide GAGKKHNNAKQSWNW (SEQ IDNO: 39) recognized CotH2 and CotH3 but not CotH1 proteins heterologouslyexpressed by S. cerevisiae. Peptide was coupled with KLH and used toraise rabbit antibodies commercially. S. cerevisiae heterologouslyexpressing CotH proteins were stained with the antibodies then counterstained with FITC labeled anti-rabbit goat antibody prior to visualizingthe cells with confocal microscopy.

FIG. 54, panels A-C, show endothelial cell surface GRP78 binds to S.cerevisiae cells heterologously expressing CotH2 or CotH3 but not CotH1and S. cerevisiae expressing CotH2 or CotH3 adhered and invadedendothelial cells or CHO cells overexpressing GRP78 but not S.cerevisiae expressing CotH1 or empty plasmid. Endothelial cell surfaceproteins were labeled with NHS-biotin and then extracted withn-octyl-β-d-glucopyranoside in PBS containing Ca²⁺ and Mg²⁺ and proteaseinhibitors. The labeled proteins (250 μg) were incubated with yeastcells (2×10⁸), then the unbound proteins were removed by extensiverinsing with PBS containing Ca²⁺ and Mg²⁺. The membrane proteins thatremained bound to the organisms were eluted with 6 M urea, separated on10% SDS-PAGE, and identified by immunoblotting with anti-GRP78 Ab (PanelA). Adherence and endocytosis (determined by differential fluorescence)assays were carried out using endothelial cells (Panel B), or CHO parentcells or those overexpressing GRP78 (Panel C) split on 12-mm glasscoverslips. * P<0.001 vs. S. cerevisiae expressing empty plasmid orCotH1 and **P<0.001 vs. CotH1 and CotH2 expression, by Wilcoxon Rank Sumtest. N=9 from three independent experiments. Data are expressed asmedian ±interquartile range.

FIG. 55, panels A and B, show anti-CotH3 Abs (raised against peptideGAGKKHNNAKQSWNW (SEQ ID NO: 39) block endothelial cell endocytosis ofand damage by R. oryzae. Adherence and endocytosis (determined bydifferential fluorescence) assays were carried out using endothelialcells split on 12-mm glass coverslips, while damage was carried outusing the 96-well plate ⁵¹Cr-release method. Endothelial cells wereincubated with 50 μg/ml anti-CotH3 or with serum from the same rabbitprior to vaccination (control) for 1 hour prior to addition of R. oryzaegermlings. Blocking of CotH3 and CotH2 (since the antibodies react toCotH2 proteins) abrogates endocytosis of R. oryzae by endothelial cells(data derived from >700 fungal cells interacting with approximately 200endothelial cells/each group/experiment, with an average of 59% cellsbeing endocytosed in the control) (Panel A) and reduces the ability ofthe fungus to cause endothelial cell damage (Panel B). *P<0.01 comparedwith pre-vaccinated serum or with no serum by Wilcoxon rank-sum test.n=6 slides per group from 3 independent experiments for endocytosis, andn=6 wells per group from 2 independent experiments for damage assay.Data are expressed as median ±interquartile range.

FIG. 56, panels A-C, show RNA-i construct targeting CotH2 and CotH3inhibits the expression of both genes, reduces CotH2 and CotH3 proteinsynthesis on the cell surface and has no effect on the growth or thepattern of germination of R. oryzae. R. oryzae was transformed with anRNA-i construct (pRNAi) targeting CotH2 and CotH3 expression or withempty plasmid. Two transformants were shown to have >80% reduction inthe expression of CotH2 and CotH3 relative to cells transformed with theempty plasmid as determined by qRT-PCR (Panel A). Flow cytometry testingusing anti-CotH antibodies demonstrated reduction in cell surfaceexpression of CotH proteins on R. oryzae cells transformed with theRNA-i construct compared to those transformed with the empty plasmid,wild type cells or negative control (i.e. wild type R. oryzae stainedwith commercial IgG instead of anti-CotH antibodies) (Panel B). The twotransformants with reduced CotH2 and CotH3 expression had similar growthrate (Panel C) as the wild type cells or cells transformed with theempty plasmid.

FIG. 57, panels A-C, show inhibition of CotH2 and CotH3 expressioncompromise the ability of R. oryzae to invade and damage endothelialcells and CHO cells overexpressing GRP78. Adherence and endocytosis(determined by differential fluorescence) assays were carried out usingendothelial cells split on 12-mm glass coverslips, while damage wascarried out using the 96-well plate ⁵¹Cr-release method. R. oryzaegermlings transformed with the RNA-i construct caused less invasion(Panel A) and damage (Panel B) to endothelial cells when compared tocells transformed with empty plasmid. Transformants with RNA-i targetingCotH2 and CotH3 caused equivalent damage to CHO cells overexpressingGRP78 when compared to CHO parent cells. In contrast, R. oryzaegermlings transformed with the empty plasmid or wild type R. oryzaecaused significantly more damage to CHO cell overexpressing GRP78 vs.CHO parent cells (Panel C). *P<0.005 compared with empty plasmid, **P<0.01 vs. wild type or empty plasmid, and

P<0.01 vs. CHO parent cells by Wilcoxon rank-sum test. n=6 slides pergroup from 3 independent experiments for endocytosis, and n=9 wells pergroup from 3 independent experiments for damage assay. Data areexpressed as median ±interquartile range.

FIG. 58, panels A-C, show inhibition of CotH2 and CotH3 expressionattenuates virulence of R. oryzae in diabetic ketoacidotic mice. Panel Ashows the survival of mice (n=10 for wild type or 9 for RNA-i or emptyplasmid transformants) infected intratracheally with one of the threestrains. Inhaled inocula were 2.4×10³, 2.8×10³, and 2.5×10³ spores, forwild type, empty plasmid, or RNA-i cells, respectively. * P<0.003 vs.wild type or empty plasmid infected mice by log Rank test. Panel B showsthe lung and brain fungal burden of diabetic ketoacidotic mice (n=9 pergroup) infected intratracheally with wild type (1.7×10³), empty plasmid(3.0×10³) or RNA-i (3.1×10³) cells. Mice were sacrificed on day +2relative to infection and their organs processed for tissue fungalburden using SYBR green assay. Data are expressed as median±interquartile range. * P<0.001 compared to wild type or empty plasmidinfected mice by Wilcoxon Rank Sum test. Panel C shows in vivoexpression of CotH genes in lungs and brains harvested from miceinfected with wild type, empty plasmid or RNA-i construct as determinedby qRT-PCR using specific primers to each of the CotH genes. Data areexpressed as mean±SD. * P<0.001 vs. wild type or empty plasmid.

FIG. 59 shows histopathological examination of lungs harvested fromdiabetic ketoacidotic mice infected with wild type or R. oryzaetransformed with empty plasmid or RNA-i. Periodic acid Schieff (PAS)stained sections demonstrating extensive hyphal elements (arrows) fromorgans collected from mice infected with wild type or R. oryzaetransformed with empty plasmid but not R. oryzae transformed with RNA-iconstruct.

FIG. 60 shows passive immunization with antiCotH antibodies raiseagainst peptide GAGKKHNNAKQSWNW (SEQ ID NO: 39) (A) or peptideMGQTNDGAYRDPTDNNK (SEQ ID NO: 40) (B) protect mice from R. oryzaeinfection. Diabetic ketoacidotic mice were given 1 mg of antiCotH IgG orpre-vaccination serum (control) 2 hr prior to infecting intratracheallywith 2.4×10³ spores of R. oryzae 99-880 (wild type). A second dose ofthe polyclonal antibody or the pre-vaccination serum was given on day +3relative to infection. * P<0.03 vs. mice receiving pre-vaccinationserum.

FIG. 61 shows the specificities of the CotH3 molecular beacon detectionof different fungal species. The amplification plot was generated inStepOnePlus Real-Time PCR machine (Applied Biosystems). The x axis isthe time from the initiation of amplification; the y axis is theincrease in fluorescence (ΔRn); threshold fluorescence is shown as thebold horizontal line (It is equal to the average plus 2 times SD for thewater negative control samples). Signals can be amplified from watersamples (0.5 ml) spiked with 10⁵ spores of R. oryzae but not Candidaalbicans or Aspergillus fumigatus.

FIG. 62 shows the sensitivity of the CotH3 molecular beacon detection ofwater samples (0.5 ml) spiked with different inocula of R. oryzae99-880. The amplification plot was generated in StepOnePlus Real-TimePCR machine (Applied Biosystems). The x axis is the time from theinitiation of amplification; the y axis is the increase in fluorescence(ΔRn); threshold fluorescence is shown as the bold horizontal line (Itis equal to the average plus 2 times SD for the water negative controlsamples).

FIG. 63 shows the sensitivity and specificity of the CotH3 molecularbeacon probe in detection of Rhizopus oryzae spores in blood. Theamplification plot was generated in StepOnePlus Real-Time PCR machine(Applied Biosystems). The x axis is the time from the initiation ofamplification; the y axis is the increase in fluorescence (ΔRn);threshold fluorescence is shown as the bold horizontal line (It is equalto the average plus 2 times SD for the water negative control samples).Blood, is inoculated control. R: R. oryzae 99-880 at different inocula(e.g. R10=R. oryzae 10 spores used to spike 350 μl of blood), A: A.fumigants, C: C. albicans each used to spike 350 μl of blood at 10⁵cells.

FIG. 64 shows the specificity of the CotH3 molecular beacon probe indetection of Rhizopus. The amplification plot was generated in aStepOnePlus Real-Time PCR machine (Applied Biosystems). The x axis isthe time from the initiation of amplification; the y axis is theincrease in fluorescence (ΔRn); threshold fluorescence is shown as thebold horizontal line (It is equal to the average plus 2 times SD for thewater negative control samples). Blood: uninoculated blood sample;99-892: R. oryzae 99-892; ATCC62417: R. microspores ATCC62417; R1000: R.oryzae 99-880. All strains were used to spike blood (350 μl) with 10³spores.

DETAILED DESCRIPTION OF THE INVENTION

The compositions and methods disclosed herein are based, at least inpart, on the identification and characterization of cell surfaceproteins that are uniquely expressed by fungi of the Mucorales order andcan facilitate binding of endothelial cells during fungal infections.Mucormycosis, which is mainly caused by Rhizopus oryzae, ischaracterized by angioinvasion and vascular thrombosis. Interactionsbetween Mucorales and endothelial cells is an important factor inestablishing a fungal condition. The recently identified GlucoseRegulated Protein 78 (GRP78), a novel host receptor that mediatesinvasion and subsequent damage of human umbilical vein endothelial cellsby R. oryzae germlings, provides a likely target ligand for R. oryzaeand other Mucorales species to bind during invasion (Liu et al., J.Clin. Invest. 120:1914-24 (2010)).

In accordance with the present invention, provided are nucleic acidsencoding Mucorales CotH polypeptides and other polypeptides disclosedherein, or functional polypeptide fragments thereof.

As used herein, the term “Mucorales CotH” refers to sub-family membersof the CotH family of proteins, wherein the Mucorales CotH proteinsinclude cell surface proteins expressed by fungi in the Mucorales orderthat are involved in the process of adherence and invasion of hostcells, such as endothelial cell. Because Mucorales CotH proteins areunique to Mucorales, the presence or absence of Mucorales CotH nucleicacid or polypeptide or changes in Mucorales CotH nucleic acid orpolypeptide expression can serve as a marker for infection by aMucorales species, for example, mucormycosis. Thus, the inventionincludes Mucorales CotH nucleic acids and/or polypeptides that can beused for screening for a fungal condition and/or for developing drugcandidates for the treatment of a fungal condition.

The term “functional,” when used herein as a modifier of an MucoralesCotH polypeptide, or polypeptide fragment thereof, refers to apolypeptide that exhibits functional characteristics similar to CotH1,CotH2 and CotH3 as disclosed herein. For example, when CotH3 or CotH2were expressed in S. cerevisiae, the S. cerevisiae cells adhere to andinvade endothelial cells or CHO cells overexpressing GRP78. Therefore,one function of Mucorales CotH is a pro-adherence and/or pro-invasionfunction. In another aspect, a functional Mucorales CotH polypeptide orfragment thereof can also include in vivo or in vitro binding to a GRP78protein, variant or fragment thereof.

The nucleic acid molecules described herein are useful for producinginvention proteins, when such nucleic acids are incorporated into avariety of protein expression systems known to those of skill in theart. In addition, such nucleic acid molecules or fragments thereof canbe labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aninvention Mucorales CotH gene or mRNA transcript in a given sample. Thenucleic acid molecules described herein, and fragments thereof, are alsouseful as primers and/or templates in a PCR reaction for amplifyinggenes encoding invention proteins described herein.

The term “nucleic acid”, also referred to as polynucleotides,encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers and can be single stranded ordouble stranded. DNA can be either complementary DNA (cDNA) or genomicDNA, and can represent the sense strand, the anti-sense strand or both.Examples of nucleic acids are RNA, cDNA, or isolated genomic DNAencoding an Mucorales CotH polypeptide. Such nucleic acids include, butare not limited to, nucleic acids comprising substantially the samenucleotide sequence as set forth in 2, 4, 6, 9, 10, 12-14, 17, 19, 21,23, 25, 27, 29 or 31. In general, a genomic sequence of the inventionincludes regulatory regions such as promoters, enhancers, and intronsthat are outside of the exons encoding a Mucorales CotH but does notinclude proximal genes that do not encode Mucorales CotH.

Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment.

The term substantially the same nucleotide sequence refers to DNA havingsufficient identity to the reference polynucleotide, such that it willhybridize to the reference nucleotide under moderately stringenthybridization conditions. In one embodiment, DNA having substantiallythe same nucleotide sequence as the reference nucleotide sequenceencodes substantially the same amino acid sequence as that set forth inany of SEQ ID NOS: 2, 4, 6, 9, 10, 12-14, 17, 19, 21, 23, 25, 27, 29 or31. In another embodiment, DNA having substantially the same nucleotidesequence as the reference nucleotide sequence has at least 65% identitywith respect to the reference nucleotide sequence. DNA havingsubstantially the same nucleotide sequence can have at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity, at least 95%identity, at least 98% identity, or at least 99% identity to thereference nucleotide sequence.

As used herein, a “modification” of a nucleic acid can also include oneor several nucleotide additions, deletions, or substitutions withrespect to a reference sequence. A modification of a nucleic acid caninclude substitutions that do not change the encoded amino acid sequencedue to the degeneracy of the genetic code. Such modifications cancorrespond to variations that are made deliberately, or which occur asmutations during nucleic acid replication.

Exemplary modifications of the recited Mucorales CotH sequences includesequences that correspond to homologs of other species, includingspecies of the Mucorales order such as A. corymbifera, A. elegans, A.rouxii, B. circina, B. multispora, C. brefeldii, C. angarensis, C.recurvatus, D. fulva, E. anomalus, H. elegans, H. assamensis, K.cordensis, M. amphibiorum, P. parasitica, P. agaricina, P. anomala, P.circinans, R. endophyticus, R. javensis, S. umbellata, S. megalocarpus,T. elegans, T. indicae-seudaticae, Z. californiensis, R. azygosporus, R.caespitosus, R. homothallicus, R. oryzae, R. microspores, R. microsporusvar. rhizopodiformis, R. schipperae, or any other species of theMucorales order disclosed herein. The corresponding Mucorales CotHsequences of Mucorales species can be determined by methods known in theart, such as by PCR or by screening genomic, cDNA or expressionlibraries.

Another exemplary modification of the invention Mucorales CotH cancorrespond to splice variant forms of the Mucorales CotH nucleotidesequence. Additionally, a modification of a nucleotide sequence caninclude one or more non-native nucleotides, having, for example,modifications to the base, the sugar, or the phosphate portion, orhaving a modified phosphodiester linkage. Such modifications can beadvantageous in increasing the stability of the nucleic acid molecule.

The invention also encompasses nucleic acids which differ from thenucleic acids shown in SEQ ID NOS: 2, 4, 6, 9, 10, 12-14, 17, 19, 21,23, 25, 27, 29 or 31, but which have the same phenotype. Phenotypicallysimilar nucleic acids are also referred to as functionally equivalentnucleic acids. As used herein, the phrase functionally equivalentnucleic acids encompasses nucleic acids characterized by slight andnon-consequential sequence variations that will function insubstantially the same manner to produce the same protein product(s) asthe nucleic acids disclosed herein. In particular, functionallyequivalent nucleic acids encode polypeptides that are the same as thoseencoded by the nucleic acids disclosed herein or that have conservativeamino acid variations. For example, conservative variations includesubstitution of a non-polar residue with another non-polar residue, orsubstitution of a charged residue with a similarly charged residue.These variations include those recognized by skilled artisans as thosethat do not substantially alter the tertiary structure of the protein.

Further provided are nucleic acids encoding Mucorales CotH polypeptidesthat, by virtue of the degeneracy of the genetic code, do notnecessarily hybridize to the invention nucleic acids under specifiedhybridization conditions. As used herein, the term degenerate refers tocodons that differ in at least one nucleotide from a reference nucleicacid, but encode the same amino acids as the reference nucleic acid.Nucleic acids encoding the invention Mucorales CotH polypeptides can becomprised of nucleotides that encode substantially the same amino acidsequence as set forth in SEQ ID NOS: 1, 3, 5, 11, 15, 16, 18, 20, 22,24, 26, 28, 30 or 32.

In one embodiment, the invention provides an isolated nucleic acidencoding a polypeptide as disclosed herein including a Mucorales CotHpolypeptide, an immunogenic fragment thereof, or a functional fragmentthereof. The invention also provides an isolated nucleic acid encoding aMucorales CotH polypeptide, an immunogenic fragment thereof, or afunctional fragment thereof, comprising a nucleic acid selected from:(a) nucleic acid encoding an amino acid sequence set forth in SEQ IDNOS: 1, 3, 5, 11, 15, 16, 18, 20, 22, 24, 26, 28, 30 or 32, or (b)nucleic acid that hybridizes to the nucleic acid of (a) under low,moderately or highly stringent conditions, wherein said nucleic acidcontiguously encodes biologically active Mucorales CotH polypeptide, or(c) nucleic acid degenerate with respect to either (a) or (b) above,wherein said nucleic acid encodes biologically active Mucorales CotHpolypeptide. In one aspect, the nucleic acid of the invention hybridizesunder highly stringent conditions.

Hybridization refers to the binding of complementary strands of nucleicacid, for example, sense:antisense strands or probe:target-nucleic acidto each other through hydrogen bonds, similar to the bonds thatnaturally occur in chromosomal DNA. Stringency levels used to hybridizea given probe with target-DNA can be readily varied by those of skill inthe art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-nucleic acid to bind a complementarynucleic acid. The hybridized nucleic acids will generally have at leastabout 60% identity, at least about 75% identity, or at least about 85%identity; or at least about 90% identity. Moderately stringentconditions are conditions equivalent to hybridization in 50% formamide,5× Denhart's solution, 5×SSPE, 0.2% SDS at 42EC, followed by washing in0.2×SSPE, 0.2% SDS, at 42EC.

The phrase “highly stringent hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65EC, for example, if a hybrid is notstable in 0.018M NaCl at 65EC, it will not be stable under highstringency conditions, as contemplated herein. High stringencyconditions can be provided, for example, by hybridization in 50%formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42EC, followed bywashing in 0.1×SSPE, and 0.1% SDS at 65EC.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution,6×SSPE, 0.2% SDS at 22EC, followed by washing in 1×SSPE, 0.2% SDS, at37EC. Denhart's solution contains 1% Ficoll, 1% polyvinylpyrolidone, and1% bovine serum albumin (BSA). 20×SSPE (sodium chloride, sodiumphosphate, ethylene diamide tetraacetic acid (EDTA)) contains 3M sodiumchloride, 0.2M sodium phosphate, and 0.025 M (EDTA). Other suitablemoderate stringency and high stringency hybridization buffers andconditions are well known to those of skill in the art and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989; and Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1999)). Nucleic acids encodingpolypeptides hybridize under moderately stringent or high stringencyconditions to substantially the entire sequence, or substantialportions, for example, typically at least 15-30 nucleotides of thenucleic acid sequence set forth in SEQ ID NOS: 2, 4, 6, 9, 10, 12-14,17, 19, 21, 23, 25, 27, 29 or 31.

The invention also provides a modification of a Mucorales CotHnucleotide sequence that hybridizes to a Mucorales CotH nucleic acidmolecule, for example, a nucleic acid molecule set forth in SEQ ID NOS:2, 4, 6, 9, 10, 12-14, 17, 19, 21, 23, 25, 27, 29 or 31, undermoderately stringent conditions. Modifications of Mucorales CotHnucleotide sequences, where the modification has at least 65% identityto a Mucorales CotH nucleotide sequence, are also provided. Theinvention also provides modification of a Mucorales CotH nucleotidesequence having at least 65% identity, at least 70% identity, at least75% identity, at least 80% identity, at least 85% identity, at least 90%identity, at least 95% identity, at least 98% identity, or at least 99%identity. The invention also provides modification of Mucorales CotHnucleotide sequences, wherein the amino acid sequence encoded by themodified nucleic acid has 65% identity, at least 70% identity, at least75% identity, at least 80% identity, at least 85% identity, at least 90%identity, at least 95% identity, at least 98% identity, or at least 99%identity to the amino acid sequence set forth in SEQ ID NOS: 1, 3, 5,11, 15, 16, 18, 20, 22, 24, 26, 28, 30 or 32.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present invention.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al., supra. Preferably, default parameters are used foralignment. One alignment program is BLAST, using default parameters. Inparticular, programs are BLASTN and BLASTP, using the following defaultparameters: Genetic code=standard; filter=none; strand=both; cutoff=60;expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGHSCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the National Center for Biotechnology Information. Biologicallyequivalent polynucleotides are those having the specified percenthomology and encoding a polypeptide having the same or similarbiological activity.

One means of isolating a nucleic acid encoding a Mucorales CotHpolypeptide is to probe a cDNA library or genomic library with a naturalor artificially designed nucleic acid probe using methods well known inthe art. Nucleic acid probes derived from the Mucorales CotH gene areparticularly useful for this purpose. DNA and cDNA molecules that encodeMucorales CotH polypeptides can be used to obtain complementary genomicDNA, cDNA or RNA from any number of Mucorales species sources, or toisolate related cDNA or genomic clones by the screening of cDNA orgenomic libraries, by methods well known in the art (see, for example,Sambrook et al., supra, 1989; Ausubel et al., supra, 1999).

The invention additionally provides a Mucorales CotH oligonucleotidecomprising between 15 and 300 contiguous nucleotides of SEQ ID NOS: 2,4, 6, 9, 10, 12-14, 17, 19, 21, 23, 25, 27, 29 or 31, or the anti-sensestrand thereof. As used herein, the term “oligonucleotide” refers to anucleic acid molecule that includes at least 15 contiguous nucleotidesfrom a reference nucleotide sequence, and can include at least 16, 17,18, 19, 20 or at least 25 contiguous nucleotides, and often includes atleast 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, up to 350 contiguous nucleotides from the referencenucleotide sequence. The reference nucleotide sequence can be the sensestrand or the anti-sense strand. Accordingly, in one aspect of theinventions, CotH oligonucleotides can comprise the nucleic acid sequenceof ATGAAATTATCTATTATATCCGCTGCC (SEQ ID NO: 33), GCTGGGAATATAATTGTCATCGA(SEQ ID NO: 34), GATGACAATTATATTCCCAGC (SEQ ID NO: 35),GAGTAGACGTAATTAGATCCAA (SEQ ID NO: 36) AAACGTACCTGCTGACCGAATC (SEQ IDNO: 37) or oligonucleotide disclosed herein.

The Mucorales CotH oligonucleotides of the invention that contain atleast 15 contiguous nucleotides of a reference Mucorales CotH nucleotidesequence are able to hybridize to Mucorales CotH under moderatelystringent hybridization conditions and thus can be advantageously used,for example, as probes to detect Mucorales CotH DNA or RNA in a sample,and to detect splice variants thereof; as sequencing or PCR primers; asantisense reagents to block transcription of Mucorales CotH RNA incells; or in other applications known to those skilled in the art inwhich hybridization to a Mucorales CotH nucleic acid molecule isdesirable.

The isolated Mucorales CotH nucleic acid molecules of the invention canbe used in a variety of diagnostic and therapeutic applications. Forexample, the isolated Mucorales CotH nucleic acid molecules of theinvention can be used as probes, as described above; as templates forthe recombinant expression of Mucorales CotH polypeptides; or inscreening assays to identify cellular molecules that bind MucoralesCotH.

Another useful method for producing a Mucorales CotH nucleic acidmolecule of the invention involves amplification of the nucleic acidmolecule using PCR and Mucorales CotH oligonucleotides and, optionally,purification of the resulting product by gel electrophoresis. Either PCRor RT-PCR can be used to produce a Mucorales CotH nucleic acid moleculehaving any desired nucleotide boundaries. Desired modifications to thenucleic acid sequence can also be introduced by choosing an appropriateoligonucleotide primer with one or more additions, deletions orsubstitutions. Such nucleic acid molecules can be amplifiedexponentially starting from as little as a single gene or mRNA copy,from any cell, tissue or species of interest.

The invention thus provides methods for detecting Mucorales CotH nucleicacid in a sample. The methods of detecting Mucorales CotH nucleic acidin a sample can be either qualitative or quantitative, as desired. Forexample, the presence, abundance, integrity or structure of a MucoralesCotH can be determined, as desired, depending on the assay format andthe probe used for hybridization or primer pair chosen for application.

Useful assays for detecting Mucorales CotH nucleic acid based onspecific hybridization with an isolated Mucorales CotH nucleic acidmolecule are well known in the art and include, for example, in situhybridization, which can be used to detect altered chromosomal locationof the nucleic acid molecule, altered gene copy number, and RNAabundance, depending on the assay format used. Other hybridizationassays include, for example, Northern blots and RNase protection assays,which can be used to determine the abundance and integrity of differentRNA splice variants, and Southern blots, which can be used to determinethe copy number and integrity of DNA. A Mucorales CotH hybridizationprobe can be labeled with any suitable detectable moiety, such as aradioisotope, fluorochrome, chemiluminescent marker, biotin, or otherdetectable moiety known in the art that is detectable by analyticalmethods.

Useful assays for detecting a Mucorales CotH nucleic acid in a samplebased on amplifying a Mucorales CotH nucleic acid with two or moreMucorales CotH oligonucleotides are also well known in the art, andinclude, for example, qualitative or quantitative polymerase chainreaction (PCR); reverse-transcription PCR (RT-PCR); single strandconformational polymorphism (SSCP) analysis, which can readily identifya single point mutation in DNA based on differences in the secondarystructure of single-strand DNA that produce an altered electrophoreticmobility upon non-denaturing gel electrophoresis; and coupled PCR,transcription and translation assays, such as a protein truncation test,in which a mutation in DNA is determined by an altered protein producton an electrophoresis gel. Additionally, the amplified Mucorales CotHnucleic acid can be sequenced to detect mutations and mutationalhot-spots, and specific assays for large-scale screening of samples toidentify such mutations can be developed.

The invention further provides an isolated Mucorales CotH polypeptides,immunogenic fragment thereof, or a functional fragment thereof, encodedby a Mucorales CotH nucleic acid of the invention. For example, theinvention provides a polypeptide comprising the same or substantiallythe same amino acid sequence as set forth in SEQ ID NOS: 1, 3, 5, 11,15, 16, 18, 20, 22, 24, 26, 28, 30 or 32. Also provided is a MucoralesCotH polypeptide encoded by a nucleotide sequence comprising the same orsubstantially the same nucleotide sequence as set forth in SEQ ID NOS:2, 4, 6, 9, 10, 12-14, 17, 19, 21, 23, 25, 27, 29 or 31.

As employed herein, the term substantially the same amino acid sequencerefers to amino acid sequences having at least about 65% identity withrespect to the reference amino acid sequence, and retaining comparablefunctional and biological activity characteristic of the protein definedby the reference amino acid sequence. In one aspect, proteins havingsubstantially the same amino acid sequence will have at least 65%identity, at least 70% identity, at least 75% identity, at least 80%identity, at least 85% identity, at least 90% identity, at least 95%identity, at least 98% identity, or at least 99% identity. It isrecognized, however, that polypeptides, or encoding nucleic acids,containing less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions, or by substitution of degenerate codons are alsoencompassed within the scope of the present invention.

Also encompassed by the term Mucorales CotH are functional fragments orpolypeptide analogs thereof. The term “functional fragment” refers to apeptide fragment that is a portion of a full length Mucorales CotHprotein, provided that the portion has a biological activity, as definedherein, that is characteristic of the corresponding full length protein.For example, in aspect of the invention, the functional fragments of theinvention can bind to the GRP78 protein or more specifically thefunctional fragments of the invention can bind to the GRP78 proteinexpressed by epithelial cells. Thus, the invention also providesfunctional fragments of invention Mucorales CotH proteins, which can beidentified using the binding and routine methods, such as bioassaysdescribed herein.

As used herein, the term “polypeptide” when used in reference toMucorales CotH is intended to refer to a peptide or polypeptide of twoor more amino acids. The term polypeptide analog includes anypolypeptide having an amino acid residue sequence substantially the sameas a sequence specifically described herein in which one or moreresidues have been conservatively substituted with a functionallysimilar residue and which displays the ability to functionally mimic aMucorales CotH as described herein. A “modification” of a Mucorales CotHpolypeptide also encompasses conservative substitutions of a MucoralesCotH polypeptide amino acid sequence. Conservative substitutions ofencoded amino acids include, for example, amino acids that belong withinthe following groups: (1) non-polar amino acids (Gly, Ala, Val, Leu, andIle); (2) polar neutral amino acids (Cys, Met, Ser, Thr, Asn, and Gln);(3) polar acidic amino acids (Asp and Glu); (4) polar basic amino acids(Lys, Arg and His); and (5) aromatic amino acids (Phe, Trp, Tyr, andHis). Other minor modifications are included within Mucorales CotHpolypeptides so long as the polypeptide retains some or all of itsfunction as described herein.

The amino acid length of functional fragments or polypeptide analogs ofthe present invention can range from about 5 amino acids up to thefull-length protein sequence of an invention Mucorales CotH. In certainembodiments, the amino acid lengths include, for example, at least about10 amino acids, at least about 15, at least about 20, at least about 25,at least about 30, at least about 35, at least about 40, at least about45, at least about 50, at least about 75, at least about 100, at leastabout 150, at least about 200, at least about 250 or more amino acids inlength up to the full-length Mucorales CotH protein sequence. Thefunctional fragments can be contiguous amino acid sequences of aMucorales CotH polypeptide, including contiguous amino acid sequences ofSEQ ID NOS: 1, 3, 5, 11, 15, 16, 18, 20, 22, 24, 26, 28, 30 or 32.

In another embodiment, the invention provides an immunogenic fragment ofthe Mucorales CotH polypeptides disclosed herein. The immunogenicfragments of the invention can include immunogenic epitopes, which canbe identified using experimental methods well known in the art.Additionally, computational modeling can also be used to identifyimmunogenic epitopes. See, for example, Tong et al. (Brief Bioinform.8(2):96-108 (2006)) and Ponomarenko et al. (2008) “B-cell epitopeprediction,” in Structural Bioinformatics, Bourne P E and Gu J (eds)Wiley-Liss; 2 edition, pgs. 849-879. Once an epitope bearing reactivitywith an antibody raised against the intact protein is identified, thepolypeptide can be tested for specificity by amino acid substitution atevery position and/or extension at both C and/or N terminal ends. Suchepitope bearing polypeptides typically contain at least six to fourteenamino acid residues, and can be produced, for example, by polypeptidesynthesis using methods well known in the art or by fragmenting anexisting protein. Accordingly, in some aspects of the invention, animmunogenic fragment of the Mucorales CotH polypeptides disclosed hereincan include the amino acid sequence GAGKKHNNAKQSWNW (SEQ ID NO: 39) orMGQTNDGAYRDPTDNNK (SEQ ID NO: 40).

With respect to the molecule used as immunogens pursuant to the presentinvention, those skilled in the art will recognize that the protein canbe truncated or fragmented without losing the essential qualities as animmunogenic vaccine. For example, a protein can be truncated to yield anN-terminal fragment by truncation from the C-terminal end withpreservation of the functional properties of the molecule as animmunogenic. Similarly, C-terminal fragments can be generated bytruncation from the N-terminal end with preservation of the functionalproperties of the molecule as an immunogenic. Other modifications inaccordance with the teachings and guidance provided herein can be madepursuant to this invention to create other polypeptide functionalfragments, immunogenic fragments, variants, analogs or derivativesthereof, to achieve the therapeutically useful properties describedherein with the native proteins.

Accordingly, the term “immunogenic fragment” as it is used herein refersto a portion of a protein that is recognized by a T-cell and/or B-cellantigen receptor. The immunogenic portion generally includes at least 5amino acid residues, or alternatively at least 6, or alternatively atleast 7, or alternatively at least 8, or alternatively at least 9, oralternatively at least 10, or alternatively at least 11, oralternatively at least 12, or alternatively at least 13, oralternatively at least 14, or alternatively at least 15, oralternatively at least 16, or alternatively at least 17, oralternatively at least 18, or alternatively at least 18, oralternatively at least 19, or alternatively at least 20, oralternatively at least 25, or alternatively at least 30, oralternatively at least 50, or alternatively at least 100 amino acidresidues of a CotH polypeptide disclosed herein. Alternatively, theimmunogenic portion can include at most 5 amino acid residues, oralternatively at most 6, or alternatively at most 7, or alternatively atmost 8, or alternatively at most 9, or alternatively at most 10, oralternatively at most 11, or alternatively at most 12, or alternativelyat most 13, or alternatively at most 14, or alternatively at most 15, oralternatively at most 16, or alternatively at most 17, or alternativelyat most 18, or alternatively at most 18, or alternatively at most 19, oralternatively at most 20, or alternatively at most 25, or alternativelyat most 30, or alternatively at most 50, or alternatively at most 100amino acid residues of a CotH polypeptide disclosed herein. In someaspects, immunogenic portions can contain a small N- and/or C-terminalfragment (e.g., 5-30 amino acids, preferably 10-25 amino acids).

A modification of a polypeptide can also include derivatives, analoguesand functional mimetics thereof, provided that such polypeptide displaysthe Mucorales CotH biological activity. For example, derivatives caninclude chemical modifications of the polypeptide such as alkylation,acylation, carbamylation, iodination, or any modification thatderivatizes the polypeptide. Such derivatized molecules include, forexample, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups can be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups can be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine can be derivatized toform N-im-benzylhistidine. Also included as derivatives or analogues arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamate, and can include amino acids that are notlinked by peptide bonds. Polypeptides of the present invention alsoinclude any polypeptide having one or more additions and/or deletions ofresidues, relative to the sequence of a polypeptide whose sequence isshown herein, so long as Mucorales CotH activity is maintained.

The invention provides an isolated Mucorales CotH polypeptides,immunogenic fragment thereof, or functional fragment thereof. Theinvention Mucorales CotH polypeptides can be isolated by a variety ofmethods well-known in the art, for example, recombinant expressionsystems, precipitation, gel filtration, ion-exchange, reverse-phase andaffinity chromatography, and the like. Other well-known methods aredescribed in Deutscher et al., Guide to Protein Purification: Methods inEnzymology, Vol. 182, (Academic Press, (1990)). Alternatively, theisolated polypeptides of the present invention can be obtained usingwell-known recombinant methods (see, for example, Sambrook et al.,supra, 1989; Ausubel et al., supra, 1999). The methods and conditionsfor biochemical purification of a polypeptide of the invention can bechosen by those skilled in the art, and purification monitored, forexample, by an immunological assay or a functional assay.

An example of the means for preparing the invention polypeptide(s) is toexpress nucleic acids encoding Mucorales CotH in a suitable host cell,such as a bacterial cell, a yeast cell, an amphibian cell such as anoocyte, or a mammalian cell, using methods well known in the art, andrecovering the expressed polypeptide, again using well-knownpurification methods, so described herein. Invention polypeptides can beisolated directly from cells that have been transformed with expressionvectors as described herein. Recombinantly expressed polypeptides of theinvention can also be expressed as fusion proteins with appropriateaffinity tags, such as glutathione S transferase (GST) or poly His, andaffinity purified. The invention polypeptide, biologically functionalfragments, and functional equivalents thereof can also be produced bychemical synthesis. For example, synthetic polypeptides can be producedusing Applied Biosystems, Inc. Model 430A or 431A automatic peptidesynthesizer (Foster City, Calif.) employing the chemistry provided bythe manufacturer.

The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified Mucorales CotHmature protein or functional polypeptide fragments thereof, alone or incombination with each other. These polypeptides or proteins can berecombinantly derived, chemically synthesized or purified from nativesources. As used herein, the term “acceptable carrier” encompasses anyof the standard pharmaceutical carriers, such as phosphate bufferedsaline solution, water and emulsions such as an oil and water emulsion,and various types of wetting agents.

The invention thus provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound selected from thegroup consisting of a Mucorales CotH polypeptide, an immunogenicfragment thereof, or a functional fragment thereof as described herein,an antisense nucleic acid as described herein or an anti-Mucorales CotHantibody as described herein. The invention additionally provides amethod of treating or preventing mucormycosis in a subject in needthereof by administering a therapeutically effective amount of apharmaceutical composition containing a pharmaceutically acceptablecarrier and a compound selected from the group consisting of a MucoralesCotH polypeptide, an immunogenic fragment thereof, or a functionalfragment thereof as described herein, an antisense nucleic acid asdescribed herein or an anti-Mucorales CotH antibody as described herein.The invention additionally provides a method of treating or preventingmucormycosis in a subject in need thereof by administering antherapeutically effective amount of a vaccine composition as disclosedherein.

Also provided are antisense-nucleic acids having a sequence capable ofbinding specifically with full-length or any portion of an mRNA thatencodes Mucorales CotH polypeptides so as to prevent translation of themRNA. The antisense-nucleic acid can have a sequence capable of bindingspecifically with any portion of the sequence of the cDNA encodingMucorales CotH polypeptides. As used herein, the phrase bindingspecifically encompasses the ability of a nucleic acid sequence torecognize a complementary nucleic acid sequence and to formdouble-helical segments therewith via the formation of hydrogen bondsbetween the complementary base pairs. An example of an antisense-nucleicacid is an antisense-nucleic acid comprising chemical analogs ofnucleotides.

The present invention provides means to modulate levels of expression ofMucorales CotH polypeptides by recombinantly expressing Mucorales CotHanti-sense nucleic acids or employing synthetic anti-sense nucleic acidcompositions (hereinafter SANC) that inhibit translation of mRNAencoding these polypeptides. Synthetic oligonucleotides, or otherantisense-nucleic acid chemical structures designed to recognize andselectively bind to mRNA are constructed to be complementary tofull-length or portions of an Mucorales CotH coding strand, includingnucleotide sequences set forth in SEQ ID NOS: 2, 4, 6, 9, 10, 12-14, 17,19, 21, 23, 25, 27, 29 or 31.

The SANC is designed to be stable in the blood stream for administrationto a subject by injection, or in laboratory cell culture conditions. TheSANC is designed to be capable of passing through the cell membrane inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SANC, which render it capable of passingthrough cell membranes, for example, by designing small, hydrophobicSANC chemical structures, or by virtue of specific transport systems inthe cell which recognize and transport the SANC into the cell. Inaddition, the SANC can be designed for administration only to certainselected cell populations by targeting the SANC to be recognized byspecific cellular uptake mechanisms which bind and take up the SANC onlywithin select cell populations. In a particular embodiment the SANC isan antisense oligonucleotide.

For example, the SANC may be designed to bind to a receptor found onlyin a certain cell type, as discussed above. The SANC is also designed torecognize and selectively bind to target mRNA sequence, which cancorrespond to a sequence contained within the sequences shown in SEQ IDNOS: 2, 4, 6, 9, 10, 12-14, 17, 19, 21, 23, 25, 27, 29 or 31. The SANCis designed to inactivate target mRNA sequence by either binding theretoand inducing degradation of the mRNA by, for example, RNase I digestion,or inhibiting translation of mRNA target sequence by interfering withthe binding of translation-regulating factors or ribosomes, or inclusionof other chemical structures, such as ribozyme sequences or reactivechemical groups which either degrade or chemically modify the targetmRNA. SANCs have been shown to be capable of such properties whendirected against mRNA targets (see Cohen et al., TIPS, 10:435 (1989) andWeintraub, Sci. American, January (1990), pp. 40).

Compositions comprising an amount of the antisense-nucleic acid of theinvention, effective to reduce expression of Mucorales CotH polypeptidesby entering a cell and binding specifically to mRNA encoding MucoralesCotH polypeptides so as to prevent translation and an acceptablehydrophobic carrier capable of passing through a cell membrane are alsoprovided herein. Suitable hydrophobic carriers are described, forexample, in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and thelike. The acceptable hydrophobic carrier capable of passing through cellmembranes may also comprise a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type.

Antisense-nucleic acid compositions are useful to inhibit translation ofmRNA encoding invention polypeptides. Synthetic oligonucleotides, orother antisense chemical structures are designed to bind to mRNAencoding Mucorales CotH polypeptides and inhibit translation of mRNA andare useful as compositions to inhibit expression of Mucorales CotHassociated genes in a tissue sample or in a subject.

The invention also provides a method for expression of a Mucorales CotHpolypeptide by culturing cells containing a Mucorales CotH nucleic acidunder conditions suitable for expression of Mucorales CotH. Thus, thereis provided a method for the recombinant production of a Mucorales CotHof the invention by expressing the nucleic acid sequences encodingMucorales CotH in suitable host cells. Recombinant DNA expressionsystems that are suitable to produce Mucorales CotH described herein arewell-known in the art (see, for example, Ausubel et al., supra, 1999).For example, the above-described nucleotide sequences can beincorporated into vectors for further manipulation. As used herein,vector refers to a recombinant DNA or RNA plasmid or virus containingdiscrete elements that are used to introduce heterologous DNA into cellsfor either expression or replication thereof.

The invention also provides vectors containing the Mucorales CotHnucleic acids of the invention. Suitable expression vectors arewell-known in the art and include vectors capable of expressing nucleicacid operatively linked to a regulatory sequence or element such as apromoter region or enhancer region that is capable of regulatingexpression of such nucleic acid. Appropriate expression vectors includethose that are replicable in eukaryotic cells and/or prokaryotic cellsand those that remain episomal or those which integrate into the hostcell genome.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a nucleic acid can be introduced into a host cell. Thevector can be used for propagation or harboring a nucleic acid or forpolypeptide expression of an encoded sequence. A wide variety of vectorsare known in the art and include, for example, plasmids, phages andviruses. Exemplary vectors can be found described in, for example,Sambrook et al., supra; Ausubel et al., supra.

Promoters or enhancers, depending upon the nature of the regulation, canbe constitutive or regulated. The regulatory sequences or regulatoryelements are operatively linked to a nucleic acid of the invention suchthat the physical and functional relationship between the nucleic acidand the regulatory sequence allows transcription of the nucleic acid.

Suitable vectors for expression in prokaryotic or eukaryotic cells arewell known to those skilled in the art (see, for example, Ausubel etal., supra, 1999). Vectors useful for expression in eukaryotic cells caninclude, for example, regulatory elements including the SV40 earlypromoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumorvirus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus(MMLV) promoter, and the like. The vectors of the invention are usefulfor subcloning and amplifying a Mucorales CotH nucleic acid molecule andfor recombinantly expressing a Mucorales CotH polypeptide. A vector ofthe invention can include, for example, viral vectors such as abacteriophage, a baculovirus or a retrovirus; cosmids or plasmids; and,particularly for cloning large nucleic acid molecules, bacterialartificial chromosome vectors (BACs) and yeast artificial chromosomevectors (YACs). Such vectors are commercially available, and their usesare well known in the art. One skilled in the art will know or canreadily determine an appropriate promoter for expression in a particularhost cell.

The invention additionally provides recombinant cells containingMucorales CotH nucleic acids of the invention. The recombinant cells aregenerated by introducing into a host cell a vector containing aMucorales CotH nucleic acid molecule. The recombinant cells aretransducted, transfected or otherwise genetically modified. Exemplaryhost cells that can be used to express recombinant Mucorales CotHmolecules include mammalian primary cells; established mammalian celllines, such as COS, CHO, HeLa, NIH3T3, HEK 293 and PC12 cells; amphibiancells, such as Xenopus embryos and oocytes; and other vertebrate cells.Exemplary host cells also include insect cells such as Drosophila, yeastcells such as Saccharomyces cerevisiae, Saccharomyces pombe, or Pichiapastoris, and prokaryotic cells such as Escherichia coli.

In one embodiment, the invention provides a vaccine composition havingan immunogenic amount of a Mucorales CotH polypeptide, an immunogenicfragment thereof or a variant of the polypeptide. The vaccinecomposition also can include an adjuvant. The formulation of the vaccinecomposition of the invention is effective in inducing protectiveimmunity in a subject by stimulating both specific humoral (neutralizingantibodies) and effector cell mediated immune responses againstMucorales CotH polypeptide. The vaccine composition of the invention isalso used in the treatment or prophylaxis of fungal infections such as,for example, mucormycosis.

The vaccine of the present invention will contain an immunoprotectivequantity of Mucorales CotH polypeptide antigens and is prepared bymethods well known in the art. The preparation of vaccines is generallydescribed in, for example, M. F. Powell and M. J. Newman, eds., “VaccineDesign (the subunit and adjuvant approach),” Plenum Press (1995); A.Robinson, M. Cranage, and M. Hudson, eds., “Vaccine Protocols (Methodsin Molecular Medicine),” Humana Press (2003); and D. Ohagan, ed.,“Vaccine Ajuvants: Preparation Methods and Research Protocols (Methodsin Molecular Medicine),” Humana Press (2000).

Mucorales CotH polypeptide, and peptide fragments or variants thereofcan include immunogenic epitopes, which can be identified using methodsknown in the art and described in, for example, Geysen et al. Proc.Natl. Acad. Sci. USA 81: 3998 (1984)). Briefly, hundreds of overlappingshort peptides, e.g., hexapeptides, can be synthesized covering theentire amino acid sequence of the target polypeptide (i.e., MucoralesCotH). The peptides while still attached to the solid support used fortheir synthesis are then tested for antigenicity by an ELISA methodusing a variety of antisera. Antiserum against Mucorales CotH proteincan be obtained by known techniques, Kohler and Milstein, Nature 256:495-499 (1975), and can be humanized to reduce antigenicity, see, forexample, U.S. Pat. No. 5,693,762, or produced in transgenic mice leavingan unrearranged human immunoglobulin gene, see, for example, U.S. Pat.No. 5,877,397. Once an epitope bearing hexapeptide reactive withantibody raised against the intact protein is identified, the peptidecan be further tested for specificity by amino acid substitution atevery position and/or extension at both C and/or N terminal ends. Suchepitope bearing polypeptides typically contain at least six to fourteenamino acid residues, and can be produced, for example, by polypeptidesynthesis using methods well known in the art or by fragmenting anMucorales CotH polypeptide. With respect to the molecule used asimmunogens pursuant to the present invention, those skilled in the artwill recognize that the Mucorales CotH polypeptide can be truncated orfragmented without losing the essential qualities as an immunogenicvaccine. For example, Mucorales CotH polypeptide can be truncated toyield an N-terminal fragment by truncation from the C-terminal end withpreservation of the functional properties of the molecule as animmunogen. Similarly, C-terminal fragments can be generated bytruncation from the N-terminal end with preservation of the functionalproperties of the molecule as an immunogen. Other modifications inaccord with the teachings and guidance provided herein can be madepursuant to this invention to create other Mucorales CotH polypeptidefunctional fragments, immunogenic fragments, variants, analogs orderivatives thereof, to achieve the therapeutically useful propertiesdescribed herein with the native protein.

The vaccine compositions of the invention further contain conventionalpharmaceutical carriers. Suitable carriers are well known to those ofskill in the art. These vaccine compositions can be prepared in liquidunit dose forms. Other optional components, e.g., pharmaceutical gradestabilizers, buffers, preservatives, excipients and the like can bereadily selected by one of skill in the art. However, the compositionscan be lyophilized and reconstituted prior to use. Alternatively, thevaccine compositions can be prepared in any manner appropriate for thechosen mode of administration, e.g., intranasal administration, oraladministration, etc. The preparation of a pharmaceutically acceptablevaccine, having due regard to pH, isotonicity, stability and the like,is within the skill of the art.

The immunogenicity of the vaccine compositions of the invention canfurther be enhanced if the vaccine further comprises an adjuvantsubstance. Various methods of achieving adjuvant effect for the vaccineare known. General principles and methods are detailed in “The Theoryand Practical Application of Adjuvants”, 1995, Duncan E. S. Stewart-Tull(ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in “Vaccines:New Generationn Immunological Adjuvants”, 1995, Gregoriadis G et al.(eds.), Plenum Press, New York, ISBN 0-306-45283-9, both of which arehereby incorporated by reference herein.

Preferred adjuvants facilitate uptake of the vaccine molecules byantigen presenting cells (APCs), such as dendritic cells, and activatethese cells. Non-limiting examples are selected from the groupconsisting of an immune targeting adjuvant; an immune modulatingadjuvant such as a toxin, a cytokine, and a mycobacterial derivative; anoil formulation; a polymer; a micelle forming adjuvant; a saponin; animmunostimulating complex matrix (ISCOM® matrix); a particle; DDA(dimethyldioctadecylammonium bromide); aluminium adjuvants; DNAadjuvants; and an encapsulating adjuvant. Liposome formulations are alsoknown to confer adjuvant effects, and therefore liposome adjuvants areincluded according to the invention.

In addition to vaccination of subjects susceptible to fungal infectionssuch as mucormycosis, the vaccine compositions of the present inventioncan be used to treat, immunotherapeutically, subjects suffering from avariety of fungal infections. Accordingly, vaccines that contain one ormore of Mucorales CotH polynucleotides, polypeptides and/or antibodycompositions described herein in combination with adjuvants, and thatact for the purposes of prophylactic or therapeutic use, are also withinthe scope of the invention. In an embodiment, vaccines of the presentinvention will induce the body's own immune system to seek out andinhibit Mucorales CotH molecules.

The term “vaccine”, as used herein, refers to a composition that can beadministered to an individual to protect the individual against aninfectious disease. Vaccines protect against diseases by inducing orincreasing an immune response in an animal against the infectiousdisease. An exemplary infectious disease amenable to treatment with thevaccines of the invention is mucormycosis. The vaccine-mediatedprotection can be humoral and/or cell mediated immunity induced in hostwhen a subject is challenged with, for example, Mucorales CotH or animmunogenic portion or fragment thereof.

The term “adjuvant” is intended to mean a composition with the abilityto enhance an immune response to an antigen generally by being deliveredwith the antigen at or near the site of the antigen. Ability to increasean immune response is manifested by an increase in immune mediatedprotection. Enhancement of humoral immunity can be determined by, forexample, an increase in the titer of antibody raised to the antigen.Enhancement of cellular immunity can be measured by, for example, apositive skin test, cytotoxic T-cell assay, ELISPOT assay for IFN-gammaor IL-2. Adjuvants are well known in the art. Exemplary adjuvantsinclude, for example, Freud's complete adjuvant, Freud's incompleteadjuvant, aluminum adjuvants, MF59 and QS21.

The term “treating” or “treatment,” as it is used herein is intended tomean an amelioration of a clinical symptom indicative of a fungalcondition. Amelioration of a clinical symptom includes, for example, adecrease or reduction in at least one symptom of a fungal condition in atreated individual compared to pretreatment levels or compared to anindividual with a fungal condition. The term “treating” also is intendedto include the reduction in severity of a pathological condition, achronic complication or an opportunistic fungal infection which isassociated with a fungal condition. Such pathological conditions,chronic complications or opportunistic infections are exemplified belowwith reference to mucormycosis. Mucormycosis and other such pathologicalconditions, chronic complications and opportunistic infections also canbe found described in, for example, Merck Manual, Sixteenth Edition,1992, and Spellberg et al., Clin. Microbio. Rev. 18:556-69 (2005).

The term “preventing” or “prevention,” as it is used herein is intendedto mean a forestalling of a clinical symptom indicative of a fungalcondition. Such forestalling includes, for example, the maintenance ofnormal physiological indicators in an individual at risk of infection bya fungus or fungi prior to the development of overt symptoms of thecondition or prior to diagnosis of the condition. Therefore, the term“preventing” includes the prophylactic treatment of individuals to guardthem from the occurrence of a fungal condition. Preventing a fungalcondition in an individual also is intended to include inhibiting orarresting the development of the fungal condition. Inhibiting orarresting the development of the condition includes, for example,inhibiting or arresting the occurrence of abnormal physiologicalindicators or clinical symptoms such as those described above and/orwell known in the art. Therefore, effective prevention of a fungalcondition would include maintenance of normal body temperature, weight,psychological state as well as lack of lesions or other pathologicalmanifestations in an individual predisposed to a fungal condition.Individuals predisposed to a fungal condition include an individual whois immunocompromised, for example, but not limited to, an individualwith AIDS, azotemia, diabetes mellitus, diabetic ketoacidosis,neutropenia, bronchiectasis, emphysema, TB, lymphoma, leukemia, orburns, or an individual undergoing chemotherapy, bone marrow-, stemcell- and/or solid organ transplantation or an individual with a historyof susceptibility to a fungal condition. Inhibiting or arresting thedevelopment of the condition also includes, for example, inhibiting orarresting the progression of one or more pathological conditions,chronic complications or susceptibility to an opportunistic infectionassociated with a fungal condition.

A “subject,” “individual” or “patient” is used interchangeably herein,and refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, rats, rabbits,simians, bovines, ovines, porcines, canines, felines, farm animals,sport animals, pets, equines, and primates, particularly humans.

The term “fungal condition” as used herein refers to fungal diseases,infection, or colonization including superficial mycoses (i.e., fungaldiseases of skin, hair, nail and mucous membranes; for example, ringwormor yeast infection), subcutaneous mycoses (i.e., fungal diseases ofsubcutaneous tissues, fascia and bone; for example, mycetoma,chromomycosis, or sporotichosis), and systemic mycoses (i.e.,deep-seated fungal infections generally resulting from the inhalation ofair-borne spores produced by causal moulds; for example, zygomycosis,aspergillosis, cryptococcosis, candidiasis, histoplasmosis,coccidiomycosis, paracoccidiomycosis, fusariosis (hyalohyphomycoses),blastomycosis, penicilliosis or sporotrichosis.

As used herein, the term “zygomycosis” is intended to mean a fungalcondition caused by fungi of the class Zygomycetes, comprised of theorders Mucorales and Entomophthorales. The Entomophthorales are causesof subcutaneous and mucocutaneous infections known asentomophthoromycosis, which largely afflict immunocompetent hosts indeveloping countries. Zygomycosis is also referred to as mucormycosisand the two terms are used interchangeably to refer to similar types offungal infections.

As used herein, the term “mucormycosis” is intended to mean a fungalcondition caused by fungi of the order Mucorales. Mucormycosis is alife-threatening fungal infection almost uniformly affectingimmunocompromised hosts in either developing or industrializedcountries. Fungi belonging to the order Mucorales are distributed intoat least six families, all of which can cause cutaneous and deepinfections. Species belonging to the family Mucoraceae are isolated morefrequently from patients with mucormycosis than any other family. Amongthe Mucoraceae, Rhizopus oryzae (Rhizopus arrhizus) is a common cause ofinfection. Other exemplary species of the Mucoraceae family that cause asimilar spectrum of infections include, for example, Rhizopusmicrosporus var. rhizopodiformis, Absidia corymbifera, Apophysomyceselegans, Mucor species, Rhizomucor pusillus and Cunninghamella spp(Cunninghamellaceae family). Mucormycosis is well known in the art andincludes, for example, rinocerebral mucormycosis, pulmonarymucormycosis, gastrointestinal mucormycosis, disseminated mucormycosis,bone mucormycosis, mediastinum mucormycosis, trachea mucormycosis,kidney mucormycosis, peritoneum mucormycosis, superior vena cavamucormycosis or external otitis mucormycosis.

Fungi belonging to the order Mucorales are currently distributed intothe families of Choanephoraceae; Cunninghamellaceae; Mucoraceae;Mycotyphaceae; Phycomycetaceae; Pilobolaceae; Saksenaeaceae;Syncephalastraceae; and Umbelopsidaceae. Each of these fungi familiesconsists of one or more genera. For example, fungi belonging to theorder Mucorales, family Mucoraceae, are further classified into thegenera of Absidia (e.g., A. corymbifera); Actinomucor (e.g., A.elegans); Amylomyces (e.g., A. rouxii); Apophysomyces; Backusella (e.g.,B. circina); Benjaminiella (e.g., B. multispora); Chaetocladium (e.g.,C. brefeldii); Circinella (e.g., C. angarensis); Cokeromyces (e.g., C.recurvatus); Dicranophora (e.g., D. fulva); Ellisomyces (e.g., E.anomalus; Helicostylum (e.g., H. elegans); Hyphomucor (e.g., H.assamensis); Kirkomyces (e.g., K. cordensis); Mucor (e.g., M.amphibiorum); Parasitella (e.g., P. parasitica); Philophora (e.g., P.agaricina); Pilaira (e.g., P. anomala); Pirella (e.g., P. circinans);Rhizomucor (e.g., R. endophyticus); Rhizopodopsis (e.g., R. javensis);Rhizopus; Sporodiniella (e.g., S. umbellata); Syzygites (e.g., S.megalocarpus); Thamnidium (e.g., T. elegans); Thermomucor (e.g., T.indicae-seudaticae); and Zygorhynchus (e.g., Z. californiensis). Thegenus Rhizopus, for example, consists of R. azygosporus; R. caespitosus;R. homothallicus; R. oryzae; R. microsporus, R. microsporus var.rhizopodiformis and R. schipperae species.

The term “immunogenic amount” as used herein refers an effective amountof a particular epitope of a polypeptide of the invention or a fragmentor variant thereof that can induce the host immune response against thepolypeptide or the infectious agent expressing the polypeptide. Thisamount is generally in the range of 20 μg to 10 mg of antigen per doseof vaccine and depends on the subject to be treated, capacity of thesubject's immune system to synthesize antibodies, and the degree ofprotection desired. The precise amount of immunogen required can becalculated by various methods such as, for example, antibody titration.The term effective amount refers to an amount of a compound orcompositions that is sufficient to provide a desired result. Thus, asused to describe a vaccine, an effective amount refers to an amount of acompound or composition (e.g., an antigen) that is sufficient to produceor elicit a protective immune response. An effective amount with respectto an immunological composition is an amount that is sufficient toelicit an immune response, whether or not the response is protective.

The “therapeutically effective amount” will vary depending on thepolypeptide, polynucleotide, antibody, antibody fragment orcompositions, the disease and its severity and the age, weight, etc., ofthe patient to be treated all of which is within the skill of theattending clinician. It is contemplated that a therapeutically effectiveamount of one or more of a polynucleotide, polypeptide, antibody,antibody fragment or composition described herein will alter a fungalpathogen penetration through and damage of endothelial cells in thepatient as compared to the absence of treatment. As such, fungalpathogenesis is decreased. A therapeutically effective amount isdistinguishable from an amount having a biological effect (a“biologically effective amount”). A polypeptide, polynucleotide,antibody, antibody fragment or compositions of the present invention mayhave one or more biological effects in vitro or even in vivo, such asreducing function of a Mucorales CotH polypetide. A biological effect,however, may not result in any clinically measurable therapeuticallyeffect as described herein as determined by methods within the skill ofthe attending clinician.

In one embodiment, nucleic acids encoding the invention Mucorales CotHpolypeptides can be delivered into mammalian cells, either in vivo or invitro using suitable vectors well-known in the art. Suitable vectors fordelivering a Mucorales CotH polypeptide, an immunogenic fragmentthereof, or a functional fragment thereof to a mammalian cell, includeviral vectors such as retroviral vectors, adenovirus, adeno-associatedvirus, lentivirus, herpesvirus, as well as non-viral vectors such asplasmid vectors. Such vectors are useful for providing therapeutic orimmunogenic amounts of a Mucorales CotH polypeptide (see, for example,U.S. Pat. No. 5,399,346, issued Mar. 21, 1995). Delivery of MucoralesCotH polypeptides or nucleic acids therapeutically can be particularlyuseful when targeted to a muscel cell, bone marrow cell or B-cell,thereby presenting the encoded mucorales CotH polypeptide fordevelopment of an immune response. Such presentation is commonly knownin the art as a DNA vaccination.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing invention nucleic acidencoding an Mucorales CotH protein into mammalian cells are well knownin the art. These viral vectors include, for example, Herpes simplexvirus vectors (Geller et al., Science, 241:1667-1669 (1988)); vacciniavirus vectors (Piccini et al., Meth. Enzymology, 153:545-563 (1987));cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzmanand S. H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1988, pp. 78-84)); Moloney murine leukemia virus vectors(Danos et al., Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988); Blaeseet al., Science, 270:475-479 (1995); Onodera et al., J. Virol.,72:1769-1774 (1998)); adenovirus vectors (Berkner, Biotechniques,6:616-626 (1988); Cotten et al., Proc. Natl. Acad. Sci. USA,89:6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991);Li et al., Human Gene Therapy, 4:403-409 (1993); Zabner et al., NatureGenetics, 6:75-83 (1994)); adeno-associated virus vectors (Goldman etal., Human Gene Therapy, 10:2261-2268 (1997); Greelish et al., NatureMed., 5:439-443 (1999); Wang et al., Proc. Natl. Acad. Sci. USA,96:3906-3910 (1999); Snyder et al., Nature Med., 5:64-70 (1999); Herzoget al., Nature Med., 5:56-63 (1999)); retrovirus vectors (Donahue etal., Nature Med., 4:181-186 (1998); Shackleford et al., Proc. Natl.Acad. Sci. USA, 85:9655-9659 (1988); U.S. Pat. Nos. 4,405,712, 4,650,764and 5,252,479, and WIPO publications WO 92/07573, WO 90/06997, WO89/05345, WO 92/05266 and WO 92/14829; and lentivirus vectors (Kafri etal., Nature Genetics, 17:314-317 (1997)).

Vectors useful for therapeutic administration of a Mucorales CotHpolypeptide of nucleic acid can contain a regulatory element thatprovides tissue specific or inducible expression of an operativelylinked nucleic acid. One skilled in the art can readily determine anappropriate tissue-specific promotor or enhancer that allows expressionof a Mucorales CotH polypeptide or nucleic acid in a desired tissue orcell. Any of a variety of inducible promoters or enhancers can also beincluded in the vector for regulatable expression of a Mucorales CotHpolypeptide or nucleic acid. Such inducible systems, include, forexample, tetracycline inducible system (Gossen & Bizard, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992); Gossen et al., Science,268:1766-1769 (1995); Clontech, Palo Alto, Calif.); metalothioneinpromoter induced by heavy metals; insect steroid hormone responsive toecdysone or related steroids such as muristerone (No et al., Proc. Natl.Acad. Sci. USA, 93:3346-3351 (1996); Yao et al., Nature, 366:476-479(1993); Invitrogen, Carlsbad, Calif.); mouse mammary tumor virus (MMTV)induced by steroids such as glucocorticoid and estrogen (Lee et al.,Nature, 294:228-232 (1981); and heat shock promoters inducible bytemperature changes.

An inducible system particularly useful for therapeutic administrationutilizes an inducible promotor that can be regulated to deliver a levelof therapeutic product in response to a given level of drug administeredto an individual and to have little or no expression of the therapeuticproduct in the absence of the drug. One such system utilizes a Gal4fusion that is inducible by an antiprogestin such as mifepristone in amodified adenovirus vector (Burien et al., Proc. Natl. Acad. Sci. USA,96:355-360 (1999). Another such inducible system utilizes the drugrapamycin to induce reconstitution of a transcriptional activatorcontaining rapamycin binding domains of FKBP12 and FRAP in anadeno-associated virus vector (Ye et al., Science, 283:88-91 (1999)). Itis understood that any combination of an inducible system can becombined in any suitable vector, including those disclosed herein. Sucha regulatable inducible system is advantageous because the level ofexpression of the therapeutic product can be controlled by the amount ofdrug administered to the individual or, if desired, expression of thetherapeutic product can be terminated by stopping administration of thedrug.

The invention additionally provides an isolated anti-Mucorales CotHantibody having specific reactivity with a Mucorales CotH polypeptide,an immunogenic fragment thereof, or functional fragment thereof. Forexample, an anti-Mucorales CotH antibody of the invention can havespecific reactivity to a polypeptide having the amino acid sequenceGAGKKHNNAKQSWNW (SEQ ID NO: 39) or MGQTNDGAYRDPTDNNK (SEQ ID NO: 40).The anti-Mucorales CotH antibody can be a monoclonal antibody or apolyclonal antibody. The invention further provides cell lines producingmonoclonal antibodies having specific reactivity with a Mucorales CotHpolypeptide, an immunogenic fragment thereof, or functional fragmentthereof.

The invention thus provides antibodies that specifically bind aMucorales CotH polypeptide. As used herein, the term “antibody” is usedin its broadest sense to include polyclonal and monoclonal antibodies,as well as antigen binding fragments of such antibodies. With regard toan anti-Mucorales CotH antibody of the invention, the term “antigen”means a native or synthesized Mucorales CotH polypeptide or fragmentthereof. An anti-Mucorales CotH antibody, or antigen binding fragment ofsuch an antibody, is characterized by having specific binding activityfor a Mucorales CotH polypeptide or a peptide portion thereof of atleast about 1×10⁵ M⁻¹. Thus, Fab, F(ab′)₂, Fd and Fv fragments of ananti-Mucorales CotH antibody, which retain specific binding activity fora Mucorales CotH polypeptide, are included within the definition of anantibody. Specific binding activity of a Mucorales CotH polypeptide canbe readily determined by one skilled in the art, for example, bycomparing the binding activity of an anti-Mucorales CotH antibody to aMucorales CotH polypeptide versus a control polypeptide that is not aMucorales CotH polypeptide. Methods of preparing polyclonal ormonoclonal antibodies are well known to those skilled in the art (see,for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press (1988)).

In addition, antibodies of the invention can be naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains as described by Huse et al. (Huseet al., Science 246:1275-1281 (1989)). These and other methods ofmaking, for example, chimeric, humanized, CDR-grafted, single chain, andbifunctional antibodies are well known to those skilled in the art(Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al.,Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard etal., Protein Engineering: A practical approach (IRL Press 1992);Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995)).

Anti-Mucorales CotH antibodies can be raised using a Mucorales CotHimmunogen such as an isolated Mucorales CotH polypeptide having theamino acid sequence of SEQ ID NOS: 1, 3, 5, 11, 15, 16, 18, 20, 22, 24,26, 28, 30 or 32, or an immunogenic fragment thereof, which can beprepared from natural sources or produced recombinantly, or a peptideportion of the Mucorales CotH polypeptide. Such peptide portions of aMucorales CotH polypeptide are functional antigenic fragments if theantigenic peptides can be used to generate a Mucorales CotH-specificantibody. A non-immunogenic or weakly immunogenic Mucorales CotHpolypeptide or portion thereof can be made immunogenic by coupling thehapten to a carrier molecule such as bovine serum albumin (BSA) orkeyhole limpet hemocyanin (KLH). Accordingly, in some aspects of theinvention, an immunogenic fragment of the CotH polypeptides disclosedherein can be conjugated to a carrier molecule, such as, but not limitedto KLH or BSA. Various other carrier molecules and methods for couplinga hapten to a carrier molecule are well known in the art (see, forexample, Harlow and Lane, supra, 1988). An immunogenic Mucorales CotHpolypeptide fragment can also be generated by expressing the peptideportion as a fusion protein, for example, to glutathione S transferase(GST), polyHis or the like. Methods for expressing peptide fusions arewell known to those skilled in the art (Ausubel et al., supra).

The invention further provides a method for detecting the presence of aMucorales organism in a sample by contacting a sample with a MucoralesCotH-specific antibody, and detecting the presence of specific bindingof the antibody to the sample, thereby detecting the presence of aMucorales CotH polypeptide in the sample. Mucorales CotH specificantibodies can be used in diagnostic methods and systems to detect thelevel of Mucorales CotH present in a sample. As used herein, the term“sample” is intended to mean any biological fluid, cell, tissue, organor portion thereof, that includes or potentially includes Mucorales CotHnucleic acids or polypeptides. The term includes samples present in anindividual as well as samples obtained or derived from the individual.For example, a sample can be a histologic section of a specimen obtainedby biopsy, or cells that are placed in or adapted to tissue culture. Asample further can be a subcellular fraction or extract, or a crude orsubstantially pure nucleic acid or protein preparation.

Mucorales CotH-specific antibodies can also be used for theimmunoaffinity or affinity chromatography purification of the inventionMucorales CotH. In addition, methods are contemplated herein fordetecting the presence of an invention Mucorales CotH protein in a cell,comprising contacting the cell with an antibody that specifically bindsto Mucorales CotH polypeptides under conditions permitting binding ofthe antibody to the Mucorales CotH polypeptides, detecting the presenceof the antibody bound to the Mucorales CotH polypeptide, and therebydetecting the presence of invention polypeptides in a cell. With respectto the detection of such polypeptides, the antibodies can be used for invitro diagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of targetMucorales CotH polypeptides in a sample include immunoassays that employa detectable antibody. Such immunoassays include, for example,immunohistochemistry, immunofluorescence, ELISA assays,radioimmunoassay, FACS analysis, immunoprecipitation, immunoblotanalysis, Pandex microfluorimetric assay, agglutination assays, flowcytometry and serum diagnostic assays, which are well known in the art(Harlow and Lane, supra, 1988; Harlow and Lane, Using Antibodies: ALaboratory Manual, Cold Spring Harbor Press (1999)).

An antibody can be made detectable by various means well known in theart. For example, a detectable marker can be directly attached to theantibody or indirectly attached using, for example, a secondary agentthat recognizes the Mucorales CotH specific antibody. Useful markersinclude, for example, radionucleotides, enzymes, binding proteins suchas biotin, fluorogens, chromogens and chemiluminescent labels.

As used herein, the terms label and indicating means in their variousgrammatical forms refer to single atoms and molecules that are eitherdirectly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturation to form afluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

Invention nucleic acids, oligonucleotides, including antisense, vectorscontaining invention nucleic acids, transformed host cells, polypeptidesand combinations thereof, as well as antibodies of the presentinvention, can be used to screen compounds to determine whether acompound functions as a potential agonist or antagonist of inventionpolypeptides. These screening assays provide information regarding thefunction and activity of invention polypeptides, which can lead to theidentification and design of compounds that are capable of specificinteraction with one or more types of polypeptides, peptides orproteins.

Thus, the invention provides methods for identifying compounds whichbind to Mucorales CotH polypeptides. The invention proteins can beemployed in a competitive binding assay. Such an assay can accommodatethe rapid screening of a large number of compounds to determine whichcompounds, if any, are capable of binding to Mucorales CotHpolypeptides. Subsequently, more detailed assays can be carried out withthose compounds found to bind, to further determine whether suchcompounds act as modulators, agonists or antagonists of inventionMucorales CotH polypeptides. Compounds that bind to and/or modulateinvention Mucorales CotH polypeptides can be used to treat a variety ofpathologies mediated by invention Mucorales CotH polypeptides.

Various binding assays to identify cellular proteins that interact withprotein binding domains are known in the art and include, for example,yeast two-hybrid screening assays (see, for example, U.S. Pat. Nos.5,283,173, 5,468,614 and 5,667,973; Ausubel et al., supra, 1999; Lubanet al., Curr. Opin. Biotechnol. 6:59-64 (1995)) and affinity columnchromatography methods using cellular extracts. By synthesizing orexpressing polypeptide fragments containing various Mucorales CotHsequences or deletions, the Mucorales CotH binding interface can bereadily identified.

In another embodiment of the invention, there is provided a bioassay foridentifying compounds which modulate the activity of invention MucoralesCotH polypeptides. According to this method, invention polypeptides arecontacted with an “unknown” or test substance, for example, in thepresence of a reporter gene construct responsive to a Mucorales CotHsignaling pathway, the activity of the polypeptide is monitoredsubsequent to the contact with the “unknown” or test substance, andthose substances which cause the reporter gene construct to be expressedare identified as functional ligands for Mucorales CotH polypeptides.Such reporter gene assays and systems are well known to those skilled inthe art (Ausubel et al., supra, 1999). In addition, a reporter geneconstrict can be generated using the promoter region of Mucorales CotHand screened for compounds that increase or decrease Mucorales CotH genepromoter activity. Such compounds can also be used to alter MucoralesCotH expression.

In accordance with another embodiment of the present invention,transformed host cells that recombinantly express invention polypeptidescan be contacted with a test compound, and the modulating effect(s)thereof can then be evaluated by comparing the Mucorales CotH-mediatedresponse, for example, via reporter gene expression in the presence andabsence of test compound, or by comparing the response of test cells orcontrol cells, to the presence of the compound.

As used herein, a compound or a signal that modulates the activity ofinvention polypeptides refers to a compound or a signal that alters theactivity of Mucorales CotH polypeptides so that the activity of theinvention polypeptide is different in the presence of the compound orsignal than in the absence of the compound or signal. In particular,such compounds or signals include agonists and antagonists. An agonistencompasses a compound or a signal that activates Mucorales CotH proteinexpression or biological activity. Alternatively, an antagonist includesa compound or signal that interferes with Mucorales CotH expression orbiological activity. Typically, the effect of an antagonist is observedas a blocking of agonist-induced protein activation. Antagonists includecompetitive and non-competitive antagonists.

Assays to identify compounds that modulate Mucorales CotH polypeptideexpression can involve detecting a change in Mucorales CotH polypeptideabundance in response to contacting the cell with a compound thatmodulates Mucorales CotH activity. Assays for detecting changes inpolypeptide expression include, for example, immunoassays with MucoralesCotH-specific Mucorales CotH antibodies, such as immunoblotting,immunofluorescence, immunohistochemistry and immunoprecipitation assays,as described above.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate Mucorales CotH activity generallyrequire comparison to a control. One type of a “control” is a cell orculture that is treated substantially the same as the test cell or testculture exposed to the compound, with the distinction that the “control”cell or culture is not exposed to the compound. Another type of“control” cell or culture can be a cell or culture that is identical tothe test cells, with the exception that the “control” cells or culturedo not express a Mucorales CotH polypeptide. Accordingly, the responseof the transfected cell to a compound is compared to the response, orlack thereof, of the “control” cell or culture to the same compoundunder the same reaction conditions.

The invention further provides a method for modulating an activitymediated by a Mucorales CotH polypeptide by contacting the MucoralesCotH polypeptide with an effective, modulating amount of an agent thatmodulates Mucorales CotH activity. The Mucorales CotH activity can be,for example, binding to GRP78. The invention additionally provides amethod of modulating the level of adhesion to a cell.

In some embodiment, the invention provides a method of detecting aMucorales CotH nucleic acid molecule in a sample. Such methods of theinvention can include the steps of contacting a sample with two or moreoligonucleotides disclosed herein, amplifying a nucleic acid molecule,and detecting the amplification. It is understood that methods foramplifying a nucleic acid are well known to one of skill in the art,which can be readily selected and applied to the methods of theinvention. For example, in some aspects, the amplification is performedusing polymerase chain reaction (PCR). In some aspects of the invention,at least one of the two or more oligonucleotides used in the method ofthe invention includes an oligonucleotide having the nucleic acidsequence of ATGAAATTATCTATTATATCCGCTGCC (SEQ ID NO: 33),GCTGGGAATATAATTGTCATCGA (SEQ ID NO: 34), GATGACAATTATATTCCCAGC (SEQ IDNO: 35), GAGTAGACGTAATTAGATCCAA (SEQ ID NO: 36), AAACGTACCTGCTGACCGAATC(SEQ ID NO: 37) or any oligonucleotide disclosed herein.

The invention further provides a method of diagnosing mucormycosisinfection in a subject by detecting the presence of a Mucorales organismin a sample from the patient. The method can include the steps of (a)providing a test sample from the subject; (b) contacting the sample withan agent that can binds a nucleic acid or a polypeptide of the inventionunder suitable conditions, wherein the conditions allow specific bindingof the agent to the nucleic acid or polypeptide; and (c) comparing theamount of the specific binding in the test sample with the amount ofspecific binding in a control sample, wherein an increased or decreasedamount of the specific binding in the test sample as compared to thecontrol sample is diagnostic of mucormycosis infection. In some aspectsof the invention, the agent is selected from the group consisting of ananti-Mucorales CotH antibody or a CotH oligonucleotide as describedherein.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems, preferably in kit form, comprising atleast one invention nucleic acid or antibody in a suitable packagingmaterial. The diagnostic kits containing nucleic acids are derived fromthe Mucorales CotH-encoding nucleic acids described herein. In oneembodiment, for example, the diagnostic nucleic acids are derived fromany of SEQ ID NOS: 2, 4, 6, 9, 10, 12-14, 17, 19, 21, 23, 25, 27, 29 or31 and can be oligonucleotides of the invention. In some aspects of theinvention, at least one oligonucleotide comprises a nucleic acidsequence selected from ATGAAATTATCTATTATATCCGCTGCC (SEQ ID NO: 33),GCTGGGAATATAATTGTCATCGA (SEQ ID NO: 34), GATGACAATTATATTCCCAGC (SEQ IDNO: 35), GAGTAGACGTAATTAGATCCAA (SEQ ID NO: 36), AAACGTACCTGCTGACCGAATC(SEQ ID NO: 37) or any oligonucleotide disclosed herein. Inventiondiagnostic systems are useful for assaying for the presence or absenceof nucleic acid encoding Mucorales CotH in either genomic DNA or mRNA.

A suitable diagnostic system includes at least one invention nucleicacid or antibody, as a separately packaged chemical reagent(s) in anamount sufficient for at least one assay. For a diagnostic kitcontaining nucleic acid of the invention, the kit will generally containtwo or more nucleic acids. When the diagnostic kit is to be used in PCR,the kit will contain at least two oligonucleotides that can serve asprimers for PCR. Those of skill in the art can readily incorporateinvention nucleic probes and/or primers or invention antibodies into kitform in combination with appropriate buffers and solutions for thepractice of the invention methods as described herein. A kit containinga Mucorales CotH antibody can contain a reaction cocktail that providesthe proper conditions for performing an assay, for example, an ELISA orother immunoassay, for determining the level of expression of aMucorales CotH polypeptide in a sample, and can contain control samplesthat contain known amounts of a Mucorales CotH polypeptide and, ifdesired, a second antibody specific for the anti-Mucorales CotHantibody.

The contents of the kit of the invention, for example, Mucorales CotHnucleic acids or antibodies, are contained in packaging material,preferably to provide a sterile, contaminant-free environment. Inaddition, the packaging material contains instructions indicating howthe materials within the kit can be employed both to detect the presenceor absence of a particular Mucorales CotH sequence or Mucorales CotHpolypeptide or to diagnose the presence of, or a predisposition for acondition associated with mucormycosis. The instructions for usetypically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLES Example I Cell Surface CotH3 Protein Facilitates Binding toHost GRP78 During Fungal Invasion of Endothelial Cells

Cell wall material was collected from supernatants of protoplasts of R.oryzae germlings. R. oryzae ligands bound to rGrp78 were isolated by FARWestern blot analysis using anti-Grp78 Ab and identified byMALDI-TOF-MS/MS analysis (FIG. 1). Briefly, protein spots of interestwere excised and sent to the UCLA W. M. Keck Proteomic Center foridentification on a Thermo LTQ-Orbitrap XL mass spectrometer (San Jose,Calif.) equipped with an Eksigent (Dublin, Calif.) NanoLiquidchromatography-1D plus system and an Eksigent autosampler. Proteinswithin the spots were in-gel tryptic digested as described by Shevchenkoet al. (Shevchenko et al. (1996). Proc Natl Acad Sci USA 93:14440-14445; Shevchenko et al., Anal. Chem., 68(5):850-8 (1996)). Theeluted peptides were loaded onto a CVC Microtech (Fontana, Calif.) 35 mmlength, 100 μm ID C18 pre-Trap column and washed for 10 min with 100%Buffer A (2% acetonitrile containing 0.1% formic acid) at a flow rate of5 μl/min. The peptides were separated on a 15 cm New Objective ProteoPepIntegraFrit column (Woburn, Mass.) using a flow rate of 300 nl/min. Thefollowing elution gradient was used: 0-15 min 0-30% Buffer B (98%acetonitrile containing 0.1% formic acid), 15-20 min 30-80% Buffer B and20-22 min 80% Buffer B. The column was then re-equilibrated for 13 minwith Buffer A. The eluting analytes were sprayed in positive mode intothe LTQ-Orbitrap MS using electrospray ionization voltage of 2300 V,capillary voltage of 45 V, tube lens of 130 V, and capillary temperatureof 200° C. Information dependent acquisition was performed where the 6most intense ions were selected in the m/z range of 300-1600 using a 60K resolution FTMS scan and subjecting them to MS-MS using broadbandcollision induced disassociation of normalized collision energy of 35and LTQ detection. Peaks were excluded from further MS-MS for a periodof 60 sec.

The resulting MS/MS spectra was searched against the Rhizopus oryzae99-880 database(http://www.broadinstitute.org/annotation/genome/rhizopus_oryzae/MultiHome.html)using the Matrix Science MASCOT Daemon search engine (Boston, Mass.).The following search parameters were used: peptide tolerance: ±10 ppm,MS/MS tolerance ±0.3 Da, maximum missed cleavages: 2, fixedmodifications: carboxymethyl (C) and variable modifications:deamidization (ND) and oxidation (M). Proteins identified within aparticular included those with a minimum of two unique peptides that areranked as number 1 and with an ion scores with a p<0.05.

Expression of the putative ligands in R. oryzae incubated withendothelial cells was detected by RT-PCR (FIG. 4). Interaction of theligand with GRP78 was confirmed by heterologously expressing the ligandin Saccharomyces cerevisiae (FIG. 6) and comparing its adherence to andinvasion of endothelial cells and CHO cells overexpressing GRP78 to S.cerevisiae transformed with empty plasmid (control) (FIG. 8).

Three of the ORF had homology to CotH family of proteins implicated inspore coat formation from several bacteria:

-   -   RO3G_05018, CotH1    -   RO3G_08029, CotH2    -   RO3G_11882, CotH3

A fourth ORF (RO3G_16295) is widely present in other pathogenic fungi,but none of these ORFs appear to encode a protein that has an identifiedfunction. As sequence comparison between the identified CotHpolypeptides and other bacterial CotH proteins shows very littlesequence identity (FIG. 17 and Table 1).

TABLE 1 Sequence similarity of Rhizopus CotH and bacterial CotH(different sizes) Flammeovirga Desulfotomaculum Bacillus Bacillusyaeyamensis reducens amyloliquefaciens cereus (ACY02060) (YP_001112853)(YP_001422883) (ZP_04217292) RO3G_05018 18.5 13.6 12.9 14.5 RO3G_0802918.6 15.8 13.8 13.6 RO3G_11882 18 14.1 13 13.6

RO3G_16295 appears to be a common protein, it's homologues (usually ˜25%identity at amino acid) can be seen from many different fungi as well asa few bacteria (FIGS. 18-26). All these proteins are not characterized.To name a few:

Talaromyces stipitatus ATCC 10500 (EED23986);

Penicillium marneffei ATCC 18224 (XP_002144175);

Aspergillus niger (XP_001392236);

Aspergillus nidulans (XP_658934);

Ustilago maydis (XP_760027);

Coccidioides immitis (XP_001243211);

Neurospora crassa (XP_956792);

Cryptococcus neoformans (XP_775558); and

Streptomyces lividans (EFD65170).

CotH3 and to a lesser extent CotH2 were expressed in R. oryzae germlingsinteracting with human umbilical vein endothelial cells (FIG. 4). CotH1was expressed by R. oryzae spores but not germlings interacting withendothelial cells (FIG. 3). Although FAR-Western analysis identified the4th ORF (RO3G_16295), this gene was not expressed by R. oryzae germlingsinteracting with endothelial cells. S. cerevisiae expressing CotH3, andto a lesser extent CotH2, specifically bound endothelial cell GRP78(FIG. 6). Heterologous expression of CotH3 in the non-adherent S.cerevisiae promoted adherence and subsequent invasion of endothelialcells and CHO cells overexpressing GRP78 (FIG. 58). A sequence alignmentbetween CotH3 polypeptides from various Mucorales species show an >90%sequence identity between the various species (FIG. 7 and Table 2)

These results show that R. oryzae invasion of endothelial cells isfacilitated by CotH3, or CotH2 binding to GRP78.

TABLE 2 Alignamaent of Nucleotide (CotH3 and other genera exons only) R.R. oryzae oryzae Mucor Absidia Cunninghamela R. CotH 3 99-880 99-89299-932 Corymbifera bertholetiae microsporus R. oryzae  100% 95.82 96.42%99.34% 97.80% 99.94 99-880 R. oryzae 95.82  100% 100.00%  96.53% 94.51%96.47% 99-892 Mucor 96.42% 100.00%   100% 96.53% 94.51% 96.47% 99-932Absidia 99.34% 96.53% 96.53%  100% 97.36% 99.39% CorymbiferaCunninghamela 97.80% 94.51% 94.51% 97.36%  100% 97.85% bertholetiae R.microsporus 99.94 96.47% 96.47% 99.39% 97.85%  100%

Example II CotH3 and CotH2 are Unique Mucorales Invasins that Bind toEndothelial Cell GRP78

R. oryzae and Culture Conditions

Several clinical Mucorales isolates were used in the experimentsdisclosed herein. For example, R. oryzae 99-880 and Mucor sp. 99-932were isolated from brain samples, whereas R. oryzae 99-892 and Rhizopussp 99-1150 were isolated from lungs samples of infected patients(samples were obtained from the Fungus Testing Laboratory, University ofTexas Health Science Center at San Antonio). Cunninghamellabertholletiae 182 is also a clinical isolate, which was a kind gift fromDr. Tomas Walsh (NIH). Lichtheimia corymbifera is also a clinicalisolate obtained from the DEFEAT Mucor clinical study (Spellberg et al.(2102), J Antimicorbiol Chemother 67(3):715-22).

Mucorales were grown on potato dextrose agar (PDA, BD Diagnostic) platesfor 3-5 days at 37° C., while A. fumigatus and C. albicans were grown onSabouraud dextrose agar (SDA) plates for 2 weeks and 48 h at 37° C.,respectively. The sporangiospores were collected in endotoxin freeDulbecco's phosphate buffered saline (PBS) containing 0.01% Tween 80,washed with PBS, and counted with a hemocytometer to prepare the finalinocula. For C. albicans, blastospores were collected in PBS aftergrowing the organisms in YPD medium [1% yeast extract (DifcoLaboratories), 2% bacto-peptone (Difco) and 2% glucose (Sigma)] at 30°C. for overnight. To form germlings, spores were incubated in liquid YPDmedium at 37° C. with shaking for 1-3 h based on the assay under study.Germlings were washed twice with RPMI 1640 without glutamine (IrvineScientific) for all assays used except for isolating the endothelialcell receptor experiments in which the germlings were washed twice withPBS (plus Ca²⁺ and Mg²⁺).

Heterologous Expression of CotH Genes in S. cerevisiae

The entire ORF of CotH1, CotH2, and CotH3 were PCR amplified from cDNAextracted from R. oryzae spores grown on PDA plates by using Phusionhigh fidelity PCR Kit (New England Biolabs) and the primers listed inTable 3. The pESC-LEU yeast dual expression vector (Stratagene) was usedto clone and express these genes under the Gall promoter. The vector wasdigested with BamHI and SalI. PCR amplified inserts from each of theCotH genes were cloned into pESC-LEU by using In-Fusion 2.0 Dry-Down PCRCloning Kit, per the manufacturer's instructions (ClontechLaboratories). The generated yeast expression vectors were independentlytransformed into yeast strain LL-20 by the polyethylene glycol-LiOAcmethod, and transformants were screened on the solid synthetic dextroseminimal medium lacking leucine. S. cerevisiae transformed with the emptyplasmid served as control.

TABLE 3 Primers used in this study Primer Sequences (SEQ ID NOS 41-64,respectively, in Primer Name order of appearance) Reaction/Use CotH1-FAAAAAACCCCGGATCCTATGAAATCCC RT-PCR and to clone CotH1 inTACTTTTTGTTGTATTC to expression vector pESC-Leu CotH1-RTCTGTTCCATGTCGACCTAGAAGAAAG RT-PCR and to clone CotH2 in AGGCAAATAAAGTGCto expression vector CotH2-F AAAAACCCCGGATCCTATGAAATTATCRT-PCR to clone CotH3 in to ACTCACTATAGTATCCTCT expression vectorCotH2-R TCTGTTCCATGTCGACTTAAAAGATAG RT-PCR to clone CotH3 in toCAGTGGCAACTAAAG expression vector CotH3-F AAAAAACCCCGGATCCTATGAAATTATRT-PCR to clone CotH3 in to CTATTATATCCGCTGCC expression vector CotH3-RTCTGTTCCATGTCGACTTAGAATACAA Detection in mucorales GGAGAGCTAAAGCGLigand#4-F AAAAAACCCCGGATCCTATGATTGCTA RT-PCR RO3G_16295 CCCCTTTTGAAALigand#4-R TCTGTTCCATGTCGACTTAAAAGAAAA RT-PCR RO3G_16295TAAAGAATGTTGCAGC CotH3-F-ORF ATGAAATTATCTATTATATCCGCTGCCDetection IN OTHER MUCORALS1.9Kb CotH3-R-ORF TTAGAATACAAGGAGAGCTAAAGCGDetection IN OTHER MUCORALS1.9Kb RNAi-cotHF-FGCATGCTAGAACAGAAGAAAGTTTTGA RNAi-forward TCGTTC RNAi-ccoHF-RGTACGACGTTCACGAATCTGTGTAGG RNAi-forward RNAi-I-FCCGCGGGACGTTCACGAATCTGTGTAG RNAi-Reverse G RNAi-I-RGCTAGCAGAACAGAAGAAAGTTTTGAT RNAi-Reverse CGTTC CotH1-FCAAACAAATGATGGGGCCTA qRT-PCR for Expression CotH1-RCGTTTTTGTTCAAGATTTACACCA qRTPCR for Expression CotH2-FCCTAATAAGGACAACGCAAACG qRT-PCR for Expression CotH2-RTTGGCAATGGCTGTGTTATC qRT-PCR for Expression CotH3-F GCCAATCCTAATGGTGAAGCqRT-PCR for Expression CotH3-R CATGAAACGGTCGAGATCAAqRT-PCR for Expression RO Actin-F AGCTCCTTTGAACCCCAAGTqRT-PCR for Expression RO Actin-R ACGACCAGAGGCATACAAGGqRT-PCR for Expression RO 18sRNA-F GCGGATCGCATGGCC qRTPCR for CFURO 18sRNA-R CCATGATAGGGCAGAAAATCG qRTPCR for CFU

Anti-CotH Antibody Production and Cell Surface Localization

Rabbit polyclonal antibodies were raised against two peptides predictedto be antigenic. The peptides GAGKKHNNAKQSWNW (SEQ ID NO: 39), andMGQTNDGAYRDPTDNNK (SEQ ID NO: 40) were coupled with KLH and used tocommercially vaccinate rabbits (ProMab Biotechnologies Inc., Richmond,Calif.). Purified IgG from the vaccinated rabbits were used to detectcell surface localization of CotH proteins on S. cerevisiae and on R.oryzae interacting with endothelial cells (Liu et al., J. Clin. Invest.,120:1914-1924 (2010)).

For localizing the CotH proteins to the cell surface of S. cerevisiae,blastospores expressing individual CotH genes were incubated first withthe anti-CotH IgG at 1:50, followed by fluorescein isothiocyanate(FITC)-labeled goat anti-rabbit IgG at 1:100. The stained cells wereimaged with Leica confocal microscope and the entire yeast cells werevisualized with differential interference contract (DIC).

For detecting the expression of the CotH proteins on R. oryzae, sporeswere germinated in YPD for 3 hours at 37° C. Germlings were stained withthe anti-CotH IgG at 1:50, followed by FITC-labeled goat anti-rabbit IgGat 1:100. A FACSCalibur (Becton Dickinson) instrument equipped with anargon laser emitting at 488 nm was used for flow cytometric analysis.Fluorescence emission was read with a 515/40 bandpass filter.Fluorescence data were collected with logarithmic amplifiers. The meanfluorescence intensities of 10⁴ events were calculated using theCELLQUEST software.

Endothelial Cells and Chinese Hamster Ovary (CHO) Cells

Endothelial cells were collected from umbilical vein endothelial cellsby the method of Jaffe et al. (Jaffe et al., J. Clin. Invest.52:2745-2756 (1973)). The cells were harvested by using collagenase andwere grown in M-199 (Gibco BRL) enriched with 10% fetal bovine serum,10% defined bovine calf serum, L-glutamine, penicillin, and streptomycin(all from Gemini Bio-Products, CA). Second-passage cells were grown toconfluency in 96-well tissue culture plates (Costar, Van Nuys, Calif.)on fibronectin (BD Biosciences). All incubations were in 5% CO₂ at 37°C. The reagents were tested for endotoxin using a chromogenic limulusamebocyte lysate assay (BioWhittaker, Inc., Walkersville, Md.), and theendotoxin concentrations were less than 0.01 IU/ml. Endothelial cellcollection was approved by Institutional Review Board at Los AngelesBiomedical Research Institute at Harbor-UCLA Medical Center. CHO cellline C.1 which was derived from parental DHFR-deficient CHO cellsengineered to overexpress GRP78s were kind gifts of Dr. Randall Kaufman(Morris et al., J. Biol. Chem., 272:4327-4334 (1997); Reddy et al., J.Biol. Chem., 278:20915-20924 (2003)).

Extraction of Endothelial Cell Membrane Proteins

Endothelial cell membrane proteins were extracted according to themethod of Isberg and Leong (Isberg and Leong, Cell 60:861-871 (1990)).Briefly, confluent endothelial cells in 100-mm diameter tissue culturedishes were rinsed twice with warm DPBS containing Ca²⁺ and Mg²⁺(PBS-CM) and then incubated with Ez-Link Sulfo-NHS-LS Biotin (0.5 mg/ml,Pierce) in PBS-CM for 12 min at 37° C. in 5% CO₂. The cells were thenrinsed extensively with cold PBS-CM and scraped from the tissue culturedishes. The endothelial cells were collected by centrifugation at 500×gfor 5 min at 4° C. and then lysed by incubation for 20 min on ice inPBS-CM containing 5.8% n-octyl-ß-D-glucopyranoside (w/v) (Cal BioChem)and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mlpepstatin A, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) (Sigma). The celldebris was removed by centrifugation at 5000×g for 5 min at 4° C. Thesupernatant was collected and centrifuged at 100,000×g for 1 h at 4° C.The concentration of the endothelial cell proteins in the resultingsupernatant was determined using Bradford method (Bio-Rad).

RNA Interference of CotH2/CotH3

Previously described RNA interference (RNAi) technology (Ibrahim et al.,Mol. Microbiol., 77:587-604 (2010)) was utilized to inhibit theexpression of CotH2 and CotH3 in R. oryzae. A 450 bp fragment commonlyshared between CotH2 and CotH3 ORF was PCR amplified and cloned as aninverted repeat under control of the Rhizopus expression vector pPdcA-Ex(Mertens et al., Archives of microbiology 186:41-50 (2006)).Additionally, an intron from the Rhizopus pdcA gene (Skory, Curr.Microbiol., 47: 59-64 (2003)) was included between repeat to serve as alinker for stabilization of the intended dsRNA structure (Nakayashiki etal., Fungal Genet. Biol., 42:275-283 (2005); Wesley et al., Plant J.,27:581-590 (2001)). The generated plasmid was transformed into R. oryzaepyrF mutant using the biolistic delivery system (Skory, Mol. Genet.Genomics 268: 397-406 (2002)) (BioRad) and transformants were selectedon minimal medium lacking uracil.

Binding of GRP78 by S. cerevisiae Expressing CotH.

S. cerevisiae cells (8×10⁸) expressing CotH1, CotH2, CotH3, or emptyplasmid were incubated for 1 h on ice with 250 μg of biotin-labeledendothelial cell surface proteins in PBS-CM plus 1.5%n-octyl-ß-D-glucopyranoside and protease inhibitors. The unboundendothelial cell proteins were washed away by three rinses with thisbuffer. The endothelial cell proteins that remained bound to the fungalcells were eluted twice with 6M urea (Fluka) and the supernatant wascombined and concentrated to appropriate volume with a Microconcentrifugal filter (10,000 MWCO, Millipore). The proteins were thenseparated on 10% SDS-PAGE, and transferred to PVDF-plus membranes (GEWater& Process Technologies). The membrane was then treated with WesternBlocking Reagent (Roche) and probed with a rabbit anti-GRP78 antibody(Abcam) followed with secondary antibodies of HRP-conjugated goatanti-rabbit IgG (Pierce), respectively. After incubation withSuperSignal West Dura Extended Duration Substrate (Pierce), the signalswere detected using a CCD camera.

Interactions of Fungi with Endothelial or CHO Cells

The number of organisms endocytosed by endothelial cells or CHO cellswas determined using a modification of a previously describeddifferential fluorescence assay (Ibrahim et al., Infect. Immun.,63:4368-4374 (1995)). Briefly, 12-mm glass coverslips in a 24-well cellculture plate were coated with fibronectin for at least 4 hrs, andseeded with endothelial or CHO cells until confluency. After washingtwice with prewarmed HBSS, the cells were then infected with 10⁵ cellsof S. cerevisiae expressing CotH or R. oryzae in RPMI 1640 medium thathas been germinated for 1 h. Following incubation for 3 h, the cellswere fixed in 3% paraformaldehyde and the cells were stained with 1%Uvitex (a kind gift from Jay Isharani, Ciba-Geigy, Greensboro, N.C.) for1 hr, which specifically binds to the chitin of fungal cell wall. Afterwashing 3 times with PBS, the coverslips were mounted on a glass slidewith a drop of ProLong Gold antifade reagent (Molecular Probes) andsealed with nail polish. The total number of cell associated organisms(i.e. germlings adhering to monolayer) was determined by phase-contrastmicroscopy. The same field was examined by epifluorescence microscopy,and the number of uninternalized germlings (which were brightlyfluorescent) was determined. The number of endocytosed organisms wascalculated by subtracting the number of fluorescent organisms from thetotal number of visible organisms. At least 400 organisms were countedin 20-40 different fields on each slide. Two slides per arm were usedfor each experiment and the experiment was performed in triplicate ondifferent days.

R. oryzae-induced endothelial or CHO cell damage was quantified by usinga chromium (⁵¹Cr) release assay (Ibrahim et al., J. Infect. Dis.,198:1083-1090 (2008)). Briefly, endothelial cells or CHO cells grown in96-well tissue culture plates containing detachable wells were incubatedwith 1 μCi per well of Na₂ ⁵¹CrO₄ (ICN, Irvine, Calif.) in M-199 medium(for endothelial cells) or Alpha minimum Eagle's medium (for CHO cells)for 16 h. On the day of the experiment, the unincorporated ⁵¹Cr wasaspirated, and the wells were washed twice with warmed Hanks' balancedsalt solution (Irvine Scientific, Irvine, Calif.). Cells were infectedwith fungal germlings (1.5×10⁵ germinated for 1 h) suspended in 150 μlof RPMI 1640 medium (Irvine Scientific) supplemented with glutamine.Spontaneous ⁵¹Cr release was determined by incubating endothelial or CHOcells in RPMI 1640 medium supplemented with glutamine without R. oryzae.After 3 h of incubation at 37° C. in a 5% CO₂ incubator, 50% of themedium was aspirated from each well and transferred to glass tubes, andthe cells were manually detached and placed into another set of tubes.The amount of ⁵¹Cr in the aspirate and the detached well was determinedby gamma counting. The total amount of ⁵¹Cr incorporated by endothelialcells in each well equaled the sum of radioactive counts per minute ofthe aspirated medium plus the radioactive counts of the correspondingdetached wells. After the data were corrected for variations in theamount of tracer incorporated in each well, the percentage of specificendothelial cell release of ⁵¹Cr was calculated by the followingformula: [(experimental release×2)−(spontaneous release×2)]/[totalincorporation−(spontaneous release×2)]. Each experimental condition wastested at least in triplicate and the experiment repeated at least once.

For antibody blocking of adherence, endocytosis, or damage caused by R.oryzae, the assays were carried out as described above except forincubating endothelial cells with 50 μg of anti-CotH antibodies(purified IgG) or with serum obtained from the same rabbit prior tovaccination with CotH3 peptide predicted to be antigenic for 1 h priorto adding R. oryzae germlings.

In Vivo Virulence Studies

For in vivo studies, ICR male mice (≥20 g) (Taconic Farms) were renderedDKA with a single i.p. injection of 190 mg/kg streptozotocin in 0.2 mlcitrate buffer 10 days prior to fungal challenge (Ibrahim et al.,Antimicrob. Agents Chemother., 47:3343-3344 (2003)). Glycosuria andketonuria were confirmed in all mice 7 days after streptozotocintreatment. Diabetic ketoacidotic mice were infected with fungal sporesby intratracheal route after sedating the mice with ketamine (66 mg/kg)and xylazine (4.8 mg/kg) with a target inoculum of 2.5×10⁵ spores. Toconfirm the inoculum, the lungs from three mice that were sacrificedimmediately following inoculation, were homogenized in PBS andquantitatively cultured on PDA plates containing 0.1% triton andcolonies were counted following a 24 h incubation period at 37° C. Theprimary efficacy endpoint was time to moribundity. In some experiments,as a secondary endpoint, fungal burden in the lungs and brains (primarytarget organs) was determined on day +2 post infection by qPCR assay aspreviously described (Ibrahim et al., Antimicrob. Agents Chemother.,49:721-727 (2005)). Values were expressed as login spore equivalent/g oftissue. Histopathological examination was carried out on sections of theharvested organs after fixing in 10% zinc formalin. The fixed organswere embedded in paraffin, and 5 mm sections were stained withhematoxylin and eosin (H&E) or Periodic acid-Schiff stains to detect R.oryzae hyphae (Ibrahim et al., J. Clin. Invest., 117:2649-2657 (2007)).

For in vivo expression of the CotH genes, lungs and brains collectedfrom mice 48 h post infection intratracheally with wild-type R. oryzae,or transformants with empty plasmid or with RNA-i construct were flashfrozen in liquid nitrogen and process for RNA extraction using a TriReagent solution (Ambion). Reverse transcription was performed withRETROscript (Ambion) using primers listed in Table 3. For quantitativeRT-PCR, SYBR green assays were performed. Constitutively expressed ACT1was used as a control for all reactions. Calculations and statisticalanalyses were performed using ABI PRISM 7000 Sequence Detection SystemUser Bulletin 2 (Applied Biosystems).

Passive Immunization

To detect if antibodies against CotH proteins protect mice frommucormycosis, diabetic ketoacidotic mice were immunized with 1 mg ofrabbit purified anti-CotH IgG raised against GAGKKHNNAKQSWNW (SEQ ID NO:39) or MGQTNDGAYRDPTDNNK (SEQ ID NO: 40) by intraperitoneal injection 2hours prior to infecting the mice intratracheally as outlined above.Control mice were infected similarly but received a similar dose fromthe same rabbit prior to vaccinating with the CotH3 peptide. Three dayspost infection a repeated dose of the antibody or the control serum(prior to vaccination) was introduced. The primary efficacy endpoint wastime to moribundity.

Statistical Analysis

Differences in CotH expression and fungi-endothelial cell interactionswere compared by the non-parametric Wilcoxon Rank Sum test. Thenon-parametric log-rank test was used to determine differences insurvival times. Comparisons with P values of <0.05 were consideredsignificant.

Results

Isolation of Putative R. oryzae Ligand(s) that Bind to Endothelial CellGRP78.

To identify the R. oryzae ligand that binds to endothelial cell GRP78,cell wall material from supernatants of protoplasts of R. oryzaegermlings were collected (Michielse et al., Mol. Genet. Genomics,271:499-510 (2004)). Incubating protoplasts in the presence of anosmotic stabilizer (e.g. sorbitol) enables regeneration of the cellwall, and during regeneration cell wall constituents are released intothe supernatant (Pitarch et al., Mol. Cell. Proteomics, 5:79-96 (2006);Pitarch et al., Electrophoresis, 20:1001-1010 (1999)). After a 2 hincubation period, (Michielse et al., Mol. Genet. Genomics, 271:499-510(2004)) protoplasts were pelleted and the supernatant was sterilized inthe presence of protease inhibitors. The supernatant was concentratedand protein concentration was measured. Negative control samples wereprocessed similarly with the exception of absence of protoplasts. FARWestern blot analysis (Wu et al., Nat. Protoc., 2:3278-3284 (2007))using recombinant human Grp78 and anti-Grp78 Ab revealed the presence of4 bands collected from the supernatant of R. oryzae protoplasts thatbound to Grp78p (FIG. 51A). These bands were excised for proteinidentification by MALDI-TOF-MS/MS analysis. Only 4 ORFs predicted to becell surface proteins were identified with GPI anchor sequence at thec-terminus, signal peptides at the N-terminus and multiple predicted N-and O-glycosylation sites. Three of the ORF (i.e. RO3G_05018,RO3G_08029, and RO3G_11882) had limited homology of 17% at the aminoacid level to CotH family of proteins implicated in spore coat formationfrom several bacteria (Giorno et al., J. Bacteriol., 189:691-705 (2007);Naclerio et al., J. Bacteriol., 178:6407 (1996)). These were named CotH1(RO3G_05018), CotH2 (RO3G_08029) and CotH3 (RO3G_11882). The fourth ORFRO3G_16295 is widely present in many fungi and some bacteria without anidentified function.

CotH2 and CotH3 are closely related to each other with 77% identity atthe amino acid level, while CotH1 is more distantly related (FIG. 51b ).The fourth ORF had an overall identity of 10% to the three CotH proteinsat the amino acid level. Upon searching the R. oryzae (delemar) 99-880genome data base, two more related ORFs were found (66% homology at theamino acid level) and were predicted to encode GPI-anchored proteins.These two ORFS (RO3G_09276; and RO3G_01139) had distant homology toCotH1, CotH2, CotH3 proteins (20-24% at the amino acid level). TheseORFs were named CotH4 and CotH5, respectively.

The possibility of the presence of this family of genes in otherMucorales known to cause human mucormycosis was also examined. Usingprimers that span the entire CotH3 ORF (1.9 kb), bands were amplifiedfrom clinical isolates including R. oryzae 99-892, Mucor sp. 99-932,Lichtheimia corymbifera, Cunninghamella bertholletiae, and Rhizomucor.Sequence analysis of these PCR-amplified bands revealed more than 90%identity at the nucleotide and predicted amino acid level with R. oryzae99-880 CotH3. Collectively, these studies show the uniqueness of CotHfamily of genes to agents of mucormycosis.

CotH2 and CotH3 are Expressed During Interaction of R. oryzae withEndothelial Cells.

Based on the results disclosed herein, it was hypothesizes that, if anyof the isolated proteins represented a fungal ligand to GRP78, then theproteins must be expressed during R. oryzae interaction with endothelialcells. Since R. oryzae binds endothelial cell GRP78 while in germlings,the expression of these four ORFs in spores or germlings were studied.All CotH genes were expressed in the spore form while only CotH3 wasexpressed in germlings of R. oryzae. Importantly, when R. oryzaegermlings were incubated with endothelial cells, both CotH2 and CotH3were expressed (FIG. 52B) with CotH3 having 16 fold and 4 fold increasecompared to CotH1 and CotH2, respectively (FIG. 52C). Finally, thefourth ORF RO3G_16295 was not expressed R. oryzae spores or germlings(FIG. 52A) or in R. oryzae germlings interacting with endothelial cells(FIG. 52B). These results showed that CotH3 and to a lesser extent CotH2are putative candidates for interacting with GRP78 during invasion ofhuman cells.

S. cerevisiae Cells Expressing CotH2 or CotH3 Bound GRP78 and Adhered toand Invaded Endothelial Cells and CHO Cells Overexpressing GRP78.

To study the role of CotH1, CotH2 and CotH3 in interacting with theGRP78 receptor, CotH2 or CotH3 were heterologously expressed in thenone-adherent none invading S. cerevisiae. The transformed yeast cellswere tested for their ability to specifically bind endothelial cellGRP78. Antibodies raised against two CotH3 peptides predicted to beantigenic and surface expressed (GAGKKHNNAKQSWNW (SEQ ID NO: 39), andMGQTNDGAYRDPTDNNK (SEQ ID NO: 40)) recognized S. cerevisiae expressingCotH3, and to a lesser extent CotH2, but not cells expressing CotH1(FIG. 53). S. cerevisiae cells expressing CotH3 primarily bound GRP78from endothelial cell membrane protein extracts. CotH2 expressing yeastcells also bound GRP78 from the same extract but S. cerevisiaeexpressing CotH1 (FIG. 54A). These results indicated that CotH3, and tolesser extent CotH2, interact with endothelial cell GRP78 duringinvasion of R. oryzae of the endothelium. To confirm this hypothesis,the ability of the transformed yeast cells to adhere to and invadeendothelial cells in vitro was examined.

Compared to empty plasmid, S. cerevisiae expressing CotH1 had noenhancement in adherence to or endocytosis (invasion) of endothelialcells. In contrast, yeast cells expressing CotH2 or CotH3 had multiplefold increase in adherence to and invasion of endothelial cells comparedto S. cerevisiae expressing CotH1 or those transformed with emptyplasmid (FIG. 54B). Importantly, cells expressing CotH3 hadsignificantly higher ability to adhere to and invade endothelial cellscompared to yeast cells expressing CotH2. To examine if this enhancedadherence to and invasion of endothelial cells was due to interactionswith GRP78, the ability of S. cerevisiae expressing CotH1, CotH2, CotH3or empty plasmid to adhere to and invade parent CHO cells were comparedto CHO cells overexpressing GRP78 (Morris et al., J. Biol. Chem.,272:4327-4334 (1997); Reddy et al., J. Biol. Chem., 278:20915-20924(2003)).

Only yeast cells expressing CotH2 or CotH3 had significant enhancementin their adhering to and invading CHO cells overexpressing GRP78 (FIG.54C). Yeast cells expressing CotH1, CotH2 or CotH3 demonstrated noincreased ability to bind to and invade parent CHO cells. Collectively,these data show that CotH3, and to a lesser extent CotH2, represents anadhesin/invasin of R. oryzae during interacting with endothelial cellGRP78.

CotH3 Protein is a R. oryzae Invasin.

Because endocytosis of the fungus was previously shown to be aprerequisite for R. oryzae to cause endothelial cell damage, (Ibrahim etal., Infect. Immun. 73:778-783 (2005); Liu et al., J. Clin. Invest.,120:1914-1924 (2010)) blocking the function or expression of CotH3 toprotect endothelial cells from R. oryzae-induced endocytosis andsubsequent damage was investigated. Endocytosis, but not adherence, ofR. oryzae germlings was abrogated by addition of rabbit anti-CotH3polyclonal antibodies, but not pre-immune serum collected from the sameanimal (FIG. 55A). The damage to endothelial cells caused by R. oryzaegermlings was reduce by >40% using anti-CotH3 antibodies (FIG. 55B)

To complement the antibody blocking studies, suppression of CotH3 andCotH2 expression was investigated to determine their impact onadherence, endocytosis, and endothelial cell damage. Using a previouslydescribed RNA-i method (Ibrahim et al., Mol. Microbiol., 77:587-604(2010)), a ˜400 bp fragment was used to suppress both genes in oneconstruct. CotH2 and CotH3 expression in two clones of R. oryzae pyrfmutant (Skory and Ibrahim, Curr. Genet. 52:23-33 (2007)) transformedwith the RNA-i construct harboring PyrF as a selection marker (i.e.Trans 2 and Trans 6) were almost entirely abrogated compared to R.oryzae pyrf mutant transformed with empty plasmid (FIG. 56A).

Next, the cell surface expression of CotH2 and CotH3 on the constructedmutants was assayed by flow cytometry using anti-CotH3 polyclonalantibodies as described herein. R. oryzae transformed with the RNA-iconstruct expressed less cell surface CotH2 and CotH3 proteins comparedto wild-type or R. oryzae transformed with the empty plasmid (FIG. 56B).These RNA-i transformants had no difference in growth rate, cell size orgermination when compared to the wild-type or empty plasmid transformedcells (FIG. 56C). However, the reduction of R. oryzae cell surfaceexpression of CotH2 and CotH3 resulted in significant reduction ofendothelial cell endocytosis of R. oryzae germlings and subsequentendothelial cell damage (FIGS. 57A and 57B). These results show thatCotH3 and CotH2 are required for maximal invasion of endothelial cellsby R. oryzae.

To further demonstrate that CotH3 and CotH2 function as invasins viabinding to GRP78, the ability of R. oryzae germlings with CotH3 andCotH2 RNA-i construct to cause damage to CHO cells overexpressing GRP78or parent CHO cells (which do not overexpress GRP78) were compared to R.oryzae transformed with the empty plasmid or wild type R. oryzae cells(Morris et al., J. Biol. Chem., 272:4327-4334 (1997); Reddy et al., J.Biol. Chem., 278:20915-20924 (2003)). As previously shown (Liu et al.,J. Clin. Invest., 120:1914-1924 (2010)), wild type R. oryzae causedconsiderably more damage to CHO cells overexpressing GRP78 when comparedto CHO parent cells. These results were further confirmed by a similarpattern of cell damage caused by R. oryzae germlings transformed withthe empty plasmid. In contrast, CHO cells overexpressing GRP78 and CHOparent cells were equally susceptible to damage caused by R. oryzaegermlings with reduced cell surface expression of CotH3 and CotH2 (FIG.57C). Collectively, these results indicate that CotH3 and CotH2 are cellsurface proteins that mediate invasion (endocytosis) of endothelialcells via binding to GRP78.

CotH2 and CotH3 are Required for Full Virulence of R. oryzae In Vivo.

Because CotH3 and CotH2 function as invasins of endothelial cells, itwas hypothesized that these two genes are critical determinants ofvirulence. To test this hypothesis, the virulence of R. oryzae withreduced cell surface expression of CotH2 and CotH3 was compared towild-type or to R. oryzae transformed with empty plasmid using anintratracheally infected diabetic ketoacidotic mouse model. Despite theinitial infection being initiated by inoculating the lungs in thismodel, the infection hematogenously disseminates to other target organssuch as the brain. Empty plasmid harboring cells were as virulent aswild type R. oryzae cells (median survival time of 3 vs. 4 days of thewild type and the empty plasmid infected mice, respectively, P=0.33). Incontrast, mice infected with the RNAi-transformant had attenuatedvirulence, which was shown by a 10 day median survival time and ⅓ of themice surviving the lethal infection (P=0.003) (FIG. 58A). Additionally,mice infected with the RNA-i transformant had significantly less fungalburden in the lungs and brains (primary and secondary target organs)when compared to the same organs recovered from mice infected with wildtype cells or those infected with the empty plasmid transformant (FIG.58B).

To further demonstrate that the attenuated virulence observed with miceinfected with R. oryzae transformed with the RNA-i construct was due toactual inhibition of CotH2 and CotH3, the pattern of in vivo expressionof these genes was assessed in fungal hyphae recovered from the mousetarget organs. CotH1 was not expressed in mice infected with wild typeR. oryzae, or R. oryzae transformed with the empty plasmid or RNA-iconstructs. In contrast, CotH2 showed a four fold and two fold increasein expression in the lungs and brains of mice infected with either thewild type R. oryzae or R. oryzae transformed with the empty plamid,receptively (FIG. 58C) compared to CotH1. Additionally, CotH3 hadsignificantly higher expression than CotH2 in the lungs, but not brains,of mice infected with wild type the empty plasmid transformant. Finally,fungal cells recovered from mice infected with R. oryzae transformedwith the RNA-i construct had no expression of any of the CotH genes(FIG. 58C). These results indicate that CotH2 and CotH3 are expressed invivo and the reduced virulence in mice infected with R. oryzaetransformed with the RNA-i is due to a lack of expression of any of theCotH genes.

To compare the severity of infection, histopathological examination ofmice organs infected with the three different strains was conducted.Lungs harvested from mice infected with R. oryzae transformed with RNA-iconstruct had normal histology compared with lungs taken from miceinfected with the wild type or R. oryzae transformed with the emptyplasmid, which had an abundance of fungal abscesses characterized byphagocyte infiltration and substantial edema (FIG. 59).

Anti-CotH3p Antibodies Protect Diabetic Ketoacidotic Mice from R. oryzaeInfection.

Because the above data showed that CotH2 and CotH3 proteins act asinvasins to mammalian cells in vitro and because CotH2 and Cot3 wererequired for full virulence of R. oryzae in the hematogenouslydisseminated murine model infection initiated by intratrachealinoculation, the use of anti-CotH3 and CotH2 antibodies raised againstpeptide GAGKKHNNAKQSWNW (SEQ ID NO: 39) or peptide MGQTNDGAYRDPTDNNK(SEQ ID NO: 40) were investigated for their protective affect againstthe disease (antibodies raised against these 2 peptides recognized S.cerevisiae expressing either CotH2 or CotH3 proteins). 1 mg of thepolyclonal antibodies was administered to diabetic ketoacidotic mice twohours prior to and three days post infecting intratracheally with R.oryzae spores. Mice receiving the anti-CotH2 and anti-CotH3 rabbit IgGhad a significantly enhanced survival time compared to mice receivingpre-vaccination serum from the same rabbit. Survival at day 21 postinfection was 44% for the mice receiving antibodies raised againstpeptide GAGKKHNNAKQSWNW (SEQ ID NO: 39) vs. 0% survival for the micereceiving the control pre-vaccination IgG (FIG. 60A). Further, Survivalat day 14 post infection was 75% for mice receiving antibodies raisedagainst peptide MGQTNDGAYRDPTDNNK (SEQ ID NO: 40) vs. 0% survival formice receiving the control pre-vaccination IgG (FIG. 60B). These resultsdemonstrate that antibodies targeting CotH proteins can be used to treatmucormycosis.

Example III Diagnostic Methods for Detecting Mucormycosis

A series of experiments were performed to determine the detectioncapability of Nucleic Acid Sequence-Based Amplification (NASBA). A NASBAprimer pair was designed to amplify a 127 bp CotH3 using Rhizopus oryzaetotal RNA as a template.

CotH3 forward primer (SEQ ID NO: 35) 5′-GATGACAATTATATTCCCAGC-3′,CotH3 reverse primer (SEQ ID NO: 36) 5′-GAGTAGACGTAATTAGATCCAA-3′,Molecular beacon probe: (SEQ ID NO: 38)5′-CGCGATCAAACGTACCTGCTGACCGAATCGATCGCG-3′

RNAs from Aspergillus fumigatus, Candida albicans, and Rhizopus oryzaewere isolated using an RNeasy® Plant Mini Kit (Qiagen) according tomanufacturer's instructions. Total RNA isolated from four different R.oryzae spores (10, 100, 1000, 10000 spores, respectively) were added tothe NASBA reactions.

To test the specificity of the molecular beacon for Rhizopus spp., RNAsisolated from C. albicans and A. fumigatus were used as controls. 300 ngtotal RNA was added to each reaction. NASBA reactions were performedwith NucliSENS EasyQ Basic kit v2 (bioMerieux by, Boxtel, NL) accordingto manufacturer's instructions. In brief, The NASBA reaction volume was20 μl (per reaction) in MicroAmp® 96-Well Reaction Plate (AppliedBiosystems) and contained 5.4 μl of sterile water, 0.4 μl of eachprimer, 0.2 μl probe, 4 μl of 5× NASBA buffer. Then 5 μl of purified RNA(300 ng) or water (when preparing no template controls) was added to thepremix. Reaction mixtures were subsequently incubated at 65° C. for 5min, cooled down to 41° C. for 5 min, after which 5 μl of enzyme mixfrom the NucliSENS EasyQ Basic kit v2 was added. This mix consisted ofcontaining T7 RNA polymerase, AMV-RT (avian myeloblastosis virus reversetranscriptase), RNase H, and BSA (bovine serum albumin). Reactions wereincubated at 41° C. for 90 min. The fluorescence signal was measuredwith StepOnePlus Real-Time PCR machine (Applied Biosystems).

Using the NASBA amplification assay described above, the CotH3 molecularbeacon primers/probe showed differential detection of fungal species,i.e. specificity for R. oryzae. Application products from samples spikedwith R. oryzae readily show amplification, whereas amplificationproducts from samples spiked with A. fumigants or C. albicans were notdetected (FIG. 61).

The CotH3 molecular beacon primers/probe showed highly sensitivedetection of R. oryzae. Fungal spores were germinated in YPD broth for 3hours at 37° C. shaker. 100 of samples containing 10 to 10⁵ of germlingswere aliquoted into 250 μl of sheep blood. Total RNA was isolated fromthe spiked blood samples with RNeasy Plant Mini Kit (Qiagen) and elutedin 30 μl elution buffer. Five microliter of the total RNA was added toeach NASBA reaction. Applification products were detected in allsamples, include samples inoculated with only 10 germlings (FIG. 62).

The CotH3 molecular beacon primers showed not only highly sensitivedetection of R. oryzae, but also a robust specificity. Fresh R. oryzae,A. fumigatus and C. albicans spores were collected, counted andaliquoted into 10 μl of YPD broth each. 350 μl of sheep blood was addedinto each tube and incubated for 24 hours at 37° C. shaker. Total RNAwas isolated with RNeasy Plant Mini Kit (Qiagen) and eluted in 30 μl inelution buffer. Five microliter of the RNA was added to each NASBAreaction. No amplification products were detected in samples inoculatedwith 10⁶ spores of A. fumigatus or C. albicans, whereas each sampleinoculated with 10 to 10⁴ spores of R. oryzae were detectable (FIG. 63).

The CotH3 molecular beacon primers/probe showed detection of multipleRhizopus species. Fresh R. oryzae (99-880 and 99-892 isolates) and R.microsporus spores (1000 spores each) were aliquoted into 10 μl of YPDbroth. 350 μl of sheep blood was added into each tube and incubated for24 hours at 37° C. shaker. Total RNA was isolated with RNeasy Plant MiniKit (Qiagen) and eluted in 30 μl in elution buffer. Five microliter ofthe RNA was added to each NASBA reaction. Not only were samplescontaining R. oryzae 99-892 spores detectable, but samples containing R.oryzae 99-880 (R1000) and R. microspores (ATCC62417) showedamplification (FIG. 64). These results show that primers designed fromCotH genes of R. oryzae can detect other strains of R. oryzae as well asother species of Rhizopus confirming the conserved nature of CotH genesamong Mucorales.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

What is claimed is:
 1. An isolated, non-naturally occurringanti-Mucorales CotH antibody, or binding fragment thereof, that (a)specifically binds a peptide having at least 87% sequence identity toamino acids 1 to 15 of SEQ ID NO: 39 or a peptide having at least 65%sequence identify to amino acids 1 to 17 of SEQ ID NO: 40, and (b)specifically binds to a CotH polypeptide on a fungus of order Mucorales.2. The antibody of claim 1, wherein the peptide sequence has a least 87%sequence identity to the amino acid sequence of SEQ ID NO: 39 or SEQ IDNO:
 40. 3. The antibody of claim 1, wherein the peptide consists of theamino acid sequence of SEQ ID NO:
 39. 4. The antibody of claim 1,wherein the peptide consists of the amino acid sequence of SEQ ID NO:40.
 5. The antibody of claim 1, wherein the antibody specifically bindsto the amino acid sequence of SEQ ID NO:
 39. 6. The antibody of claim 1,wherein the antibody specifically binds to the amino acid sequence ofSEQ ID NO:
 40. 7. The antibody of claim 1, wherein the fungus is apathogenic fungus that can cause mucormycosis in a human.
 8. Theantibody of claim 7, wherein the pathogenic fungus belongs to the familyMucoraceae.
 9. The antibody of claim 8, wherein the pathogenic fungusbelongs to genus Rhizopus, Rhizomucor, Lichtheimia, Apophysomyces orMucor.
 10. The antibody of claim 9, wherein the pathogenic fungusbelongs to the genus Lichtheimia or Mucor.
 11. The antibody of claim 7,wherein the pathogenic fungus is a species selected from Lechtheimiacorymbifera, Cunninghamella bertholletia, Rhizopus microspores, Mucorracemosus, Mucor circinelloides, Rhizomucor variabilis, Apophysomyceselegans, and Apophysomyces trapeziformis.
 12. The antibody of claim 1,wherein the antibody is a monoclonal antibody.
 13. The antibody of claim12, wherein the monoclonal antibody is a humanized antibody.
 14. Theantibody of claim 12, wherein the monoclonal antibody is a chimericantibody.
 15. The antibody of claim 1, wherein the binding fragment is asingle chain antibody.
 16. The antibody of claim 1, wherein the antibodycomprises a detectable marker.
 17. The antibody of claim 1, wherein theantibody abrogates endocytosis of the fungus by a mammalian endothelialcell.
 18. An isolated anti-Mucorales CotH antibody, or binding fragmentthereof, that (a) specifically binds to a peptide having at least 87%sequence identity to amino acids 1 to 15 of SEQ ID NO: 39 or amino acids1 to 17 of SEQ ID NO: 40, (b) specifically binds to a CotH polypeptideon a cell surface of a fungus of genus Rhizopus, Rhizomucor,Lichtheimia, Mucor or Cunninghamella, wherein the CotH polypeptide onsaid cell surface comprises the peptide, and (c) abrogates endocytosisof the fungus by a mammalian endothelial cell, wherein (i) the antibodyis a humanized antibody, a chimeric antibody, a CDR-grafted antibody, ora bifunctional antibody, or (ii) the binding fragment is selected from asingle chain antibody, Fab, F(ab′)2, Fd and an Fv fragment.
 19. Theantibody of claim 1, wherein the non-naturally occurring antibody, orbinding fragment thereof, is selected from a humanized antibody,chimeric antibody, CDR-grafted antibody, a bifunctional antibody, asingle chain antibody, Fab, F(ab′)2, Fd and an Fv fragment.
 20. Theantibody of claim 1, wherein the fungus of order Mucorales is apathogenic fungus of family Cunninghamellaceae.